Formulations with reduced degradation of polysorbate

ABSTRACT

The invention provides methods for making such formulations and methods of using such formulations. The invention further provides methods of reducing polysorbate degradation, methods of reducing the amount of visible and sub-visible particles in an aqueous formulation, and methods of disaggregating polysorbate degradation products comprising adding a cyclodextrin to a formula comprising polysorbate and a polypeptide. The invention also provides aqueous formulations comprising a polypeptide, a polysorbate, and a cyclodextrin with reduced polysorbate degradation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/393,147, filed on Dec. 28, 2016, which claims the benefit of U.S.Provisional Application No. 62/272,965, filed Dec. 30, 2015, thecontents of which are hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to aqueous pharmaceutical formulations comprisinga cyclodextrin and a polysorbate and methods for reducing polysorbatedegradation and for disaggregating and solubilizing polysorbatedegradation products.

BACKGROUND OF THE INVENTION

Pharmaceutical formulations commonly contain polysorbates 20 and 80(PS20 and PS80), non-ionic surfactants composed of a hydrophilicpolyoxyethylene head group and a hydrophobic fatty acid tail. Theaddition of surfactants to formulations protects proteins fromsurface-induced denaturation and aggregation (Geisen, Diabetologia27:212-218 (1984); Wang, Int. J. Pharm. 289:1-30 (2005)). Proteinaggregation can occur during drug substance (DS) and drug product (DP)processing, long-term storage, shipment, and during administration(Cromwell et al., AAPS J. 8:E572-E579 (2006)). It has been shown thatthe addition of a surfactant (e.g., PS20) can minimize interfacialinteractions that may stress proteins during filtration (Maa et al., J.Pharm. Sci. 87:808-812 (1998); Maa et al., Biotechnol. Bioeng.50:319-328 (1996)), agitation (Liu et al., J. Pharm. Sci. 102:2460-2470(2013)), freeze-thaw (Kreilgaard et al., J. Pharm. Sci. 87:1597-1603(1998); Hillgren et al., Int. J. Pharm. 237:57-69 (2002)),lyophilization (Carpenter, Protein Sci. 13:54-54 (2004); Carpenter etal., Pharm. Res. 14:969-975 (1997)), reconstitution (Webb et al., J.Pharm. Sci. 91:543-558 (2002)), administration (Kumru et al., J. Pharm.Sci. 101:3636-3650 (2012)), and storage.

To ensure stabilization of active pharmaceutical ingredients (API)during processing, long-term storage, and during administration, it isimportant to prevent polysorbate degradation. However, PS20 issusceptible to degradation via hydrolytic and oxidative pathways (Kumru,et al., J. Pharm. Sci. 101:3636-3650 (2012); Mahler et al., Abstr Pap AmChem S. 239: (2010)).

Oxidative degradation of polysorbates has been well characterized andhas been studied extensively (Kerwin, J. Pharm. Sci. 97:2924-2935(2008); Kishore et al., J. Pharm. Sci. 100:721-731 (2011)). Oxidationtypically occurs in the context of two mechanisms (1) the autoxidationof the ethylene oxide group and (2) radical oxidation at the site ofunsaturation (Kishore et al., J. Pharm. Sci. 100:721-731 (2011)).Although oxidative degradation of polysorbates has been observed, it hasbeen shown that PS20 oxidation can be mitigated in protein formulationsby coformulating with antioxidants (e.g., methionine). Formulationscontaining tryptophan have also been developed to prevent oxidation ofamino acid residues (US2014/0322203; US2014/0314) Oxidative andhydrolytic polysorbate degradation pathways are distinguishable byunique degradation product profiles. Hydrolytic polysorbate degradationproduces predominantly fatty acids and oxidative polysorbate degradationproduces more diverse degradation products including peroxides,aldehydes, acids, keytones, n-alkanes, fatty acid esters, and otherdegradation products (Ravuri et al., Pharm. Res. 28:1194-1210 (2011)).

Stress models for oxidative polysorbate degradation using2,2′-Azobisisobutyramidinium (AAPH) that degrade PS20 have beendescribed previously (Borisov et al., J. Pharm. Sci. 104:1005-1018(2015)). Using similar approaches, representative stress models can beused to develop formulations that reduce oxidative polysorbatedegradation under relevant conditions.

Stress models for hydrolysis using purified esterases (e.g., PorcineLiver Esterase, etc.) and lipases (e.g., tweenase, etc.) have beendescribed previously (Labrenz, J. Pharm. Sci. 103L2268-2277 (2014)).Using similar approaches, representative stress models can be used todevelop formulations that reduce catalytic polysorbate degradation underrelevant conditions.

Recently, there have been reports of enzymatic degradation ofpolysorbate in monoclonal antibody (mAb) formulations. For example,Labrenz attributed polysorbate 80 (PS80) degradation observed inCHO-derived mAb formulations to specific enzymatic mechanism rather thana general biologic hydrolysis mechanism based on the PS20 degradationprofile (Labrenz et al., J. Pharm. Sci. 103:2268-2277 (2014)).Sequencing of the CHO cell genome has identified various host cellproteins (HCPs) (e.g., lipases) capable of degrading polysorbate (S.Hammond et al., Biotech. Bioeng. 109:1353-1356 (2012)). Subsequently,Lee et al. have shown that reducing the expression of specific HCPssubstantially reduced the hydrolysis of PS80 relative to controlsamples. These recent findings establish that lipases associated withbiologics manufacturing are expressed in upstream processes. Downstreampurification processes (e.g., Protein A) are capable of removal of HCPs;however, it has been shown that some HCPs can be co-purified with APImolecules that have similar properties and are thus retained in tracequantities in the drug substance and drug product (K. Lee, et al., AChinese Hamster Ovary Cell Host Cell Protein That Impacts PS-80Degradation. AccBio Conference (2015). Presumably, lipases with highactivity can result in significant polysorbate degradation even atundetectable levels. There are numerous ongoing efforts to identify andremove lipases from protein drugs by engineering cells with reducedlipase expression and via downstream processing steps (e.g.,chromatography). However, the enzymatic degradation of PS20 and PS80remains a significant challenge in biopharmaceutical development andthere have been no significant efforts reported to identify optimalformulations for reducing hydrolytic or catalytic PS20 degradation.

Polysorbate degradation has numerous consequences that may impact thestability and shelf-life of protein drug formulations. Polysorbatedegradants include poorly soluble fatty acids that may result in theformation of visible and subvisible particles in the solution. The lossof PS20 may also reduce the protective effects of PS20 for proteinformulations. Additionally, a spiking study demonstrated that some ofthe PS20-related degradants can impact stability of protein drugs;however, no impact was observed under pharmaceutically relevantconditions (Kishore et al., Pharm. Res. 28:1194-1210 (2011).

What is needed is a method of reducing polysorbate degradation so thatthe protective effects of polysorbate on formulations (e.g.,polypeptides) are maintained over time. This will result in more stablepolypeptide formulations during processing, long-term storage, andduring administration which in turn will lengthen the shelf life ofpolypeptide formulations and reduce waste caused by degraded and expiredformulations.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY

The invention provides a method of reducing polysorbate degradation inan aqueous formulation comprising a polysorbate, the method comprisingadding a cyclodextrin to the formulation, wherein the resulting w/wratio of cyclodextrin to polysorbate is greater than about 37.5:1. Insome aspects, the invention provides a method of reducing polysorbatedegradation in an aqueous formulation comprising a polysorbate, themethod comprising adding a cyclodextrin to the formulation wherein theresulting w/w ratio of cyclodextrin to polysorbate is greater than about37.5:1, wherein the formulation comprises about 0.005%-0.4% polysorbate.In some aspects, the invention provides a method of reducing polysorbatedegradation in an aqueous formulation comprising a polysorbate, themethod comprising adding a cyclodextrin to the formulation to aconcentration of about 0.01%-30%, wherein the resulting w/w ratio ofcyclodextrin to polysorbate is greater than about 37.5:1, wherein theformulation comprises about 0.005%-0.4% polysorbate. In some aspects,the invention provides a method of reducing the amount of sub-visibleand visible particles in an aqueous formulation comprising polysorbate,comprising adding a cyclodextrin to the formulation, wherein theresulting w/w ratio of cyclodextrin to polysorbate is greater than about37.5:1, wherein the formulation comprises polysorbate and a polypeptide.In some aspects the invention provides a method to disaggregate andsolubilize polysorbate degradation products in an aqueous formulationcomprising adding a cyclodextrin to the formulation, wherein theresulting w/w ratio of cyclodextrin to polysorbate is greater than about37.5:1, wherein the formulation comprises polysorbate and a polypeptide.

In some embodiments of the above aspects, the polysorbate is polysorbate20 or polysorbate 80. In some embodiments, the cyclodextrin isHP-βcyclodextrin, HP-γcyclodextrin, or sulfobutyl ether β-cyclodextrin.In some embodiments, the concentration of polysorbate in the formulationis in the range of about 0.01% to 0.4%. In some embodiments, theconcentration of polysorbate in the formulation is in the range of about0.01% to 0.1%. In some embodiments, the concentration of polysorbate inthe formulation is about 0.02%. In some embodiments, the concentrationof cyclodextrin in the formulation is in the range of about 0.5-30%. Insome embodiments, the concentration of cyclodextrin in the formulationis about 15%.

In some embodiments of the above aspects and embodiments, thepolysorbate degradation is reduced by about 50%, about 75%, about 80%,about 85%, about 90%, about 95% or about 99%. In some embodiments, lessthan about 1,000, about 750, about 500, about 250, about 150, about 100,about 50, or about 25 polysorbate particles greater than about 2 micronsin diameter/mL are formed.

In some embodiments of the above aspects and embodiments, theformulation comprises a polypeptide. In some embodiments, thepolypeptide is an antibody. In some embodiments, the antibody is apolyclonal antibody, a monoclonal antibody, a humanized antibody, ahuman antibody, a chimeric antibody, a multispecific antibody orantibody fragment. In some embodiments, the polypeptide concentration inthe formulation is about 1 mg/mL to about 250 mg/mL.

In some embodiments of the above aspects and embodiments, theformulation is stable at about 2° C. to about 8° C. for at least aboutsix months, at least about 12 months, at least about 18 months, or atleast about 24 months. In some embodiments, the formulation is stable atabout 1° C. to about 10° C. for at least about forty-eight months. Insome embodiments, the formulation is stable at about 2° C. to about 8°C. for at least about forty-eight months.

In some embodiments, the formulation has a pH of about 4.5 to about 7.0.In some embodiments, the formulation has a pH of about 4.5 to about 6.0.In some embodiments, the formulation has a pH of about 6.0.

In some embodiments of the above aspects and embodiments, theformulation further comprises one or more excipients selected from thegroup consisting of a stabilizer, a buffer, a surfactant, and a tonicityagent. In some embodiments, the formulation is a pharmaceuticalformulation suitable for administration to a subject. In someembodiments, the formulation is pharmaceutical formulation suitable forintravenous, subcutaneous, intramuscular, or intravitreal administrationto a subject.

In some aspects, the invention provides an aqueous formulationcomprising a polypeptide, a polysorbate and a cyclodextrin, wherein theformulation has been stored at about 1° C. to about 10° C. for at leastabout six months, wherein the initial w/w ratio of cyclodextrin topolysorbate in the formulation is at least about 37.5:1 and wherein theamount of polysorbate in the formulation is at least about 80% of theinitial amount of polysorbate in the formulation. In some aspects, theinvention provides an aqueous formulation comprising a polypeptide, apolysorbate and a cyclodextrin, wherein the formulation has been storedat about 1° C. to about 10° C. for at least about six months, whereinthe w/w ratio of cyclodextrin to polysorbate in the formulation is atleast about 37.5:1 and wherein less than about 1% of the polysorbate hasdegraded.

In some embodiments of the above aspects, the cyclodextrin isHP-βcyclodextrin, HP-γcyclodextrin, or sulfobutyl ether β-cyclodextrin.In some embodiments, the concentration of polysorbate in the formulationis in the range of about 0.01% to 0.4%. In some embodiments, theconcentration of polysorbate in the formulation is in the range of about0.01% to 0.1%. In some embodiments, the concentration of polysorbate inthe formulation is about 0.02%. In some embodiments, the concentrationof cyclodextrin in the formulation is in the range of about 0.5-30%. Insome embodiments, the concentration of cyclodextrin in the formulationis about 15%.

In some embodiments of the above aspects and embodiments, thepolysorbate degradation is reduced by about 50%, about 75%, about 80%,about 85%, about 90%, about 95% or about 99%. In some embodiments, lessthan about 1,000, about 750, about 500, about 250, about 150, about 100,about 50, or about 25 polysorbate particles greater than about 2 micronsin diameter/mL are formed.

In some embodiments of the above aspects and embodiments, theformulation is stable at about 2° C. to about 8° C. for at least aboutsix months. In some embodiments, the formulation is stable at about 1°C. to about 10° C. for at least about forty-eight months. In someembodiments, the formulation is stable at about 2° C. to about 8° C. forat least about forty-eight months.

In some embodiments of the above aspects and embodiments, theformulation further comprises a polypeptide. In some embodiments, thepolypeptide is an antibody. In some embodiments, the antibody is apolyclonal antibody, a monoclonal antibody, a humanized antibody, ahuman antibody, a chimeric antibody, a multispecific antibody orantibody fragment. the polypeptide concentration in the formulation isabout 1 mg/mL to about 250 mg/mL.

In some embodiments of the above aspects and embodiments, theformulation has a pH of about 4.5 to about 7.0. In some embodiments, theformulation has a pH of about 4.5 to about 6.0. In some embodiments, theformulation has a pH of about 6.0.

In some embodiments of the above aspects and embodiments, theformulation further comprises one or more excipients selected from thegroup consisting of a stabilizer, a buffer, a surfactant, and a tonicityagent. In some embodiments, the formulation is a pharmaceuticalformulation suitable for administration to a subject. In someembodiments, the formulation is pharmaceutical formulation suitable forintravenous, subcutaneous, intramuscular, or intravitreal administrationto a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays the average (n=3) relative percent of PS20 determined byRP-ELSD for samples oxidized with 5 mM AAPH at 40° C. for 24 hourscontaining no excipient (control), 15% (w/v) sucrose, and 15% (w/v)HP-β-CD.

FIG. 2 displays the average (n=3) relative percent of PS20 determined byRP-ELSD for samples digested using Candida Antartica Lipase B (black),Lipoprotein Lipase (grey), and Rabbit Liver Esterase (white) enzymes inprotein-free samples containing both 0.02% (w/v) PS20 with 0 and 15%(w/v) HP-β-CD.

FIGS. 3A-3D display the average (n=3) ≥2 μM (FIG. 3A), ≥5 μM (FIG. 3B),≥10 μM (FIG. 3C), and ≥25 μM (FIG. 3D) particle counts per milliliterdetermined by HIAC for samples digested using Candida Antarctica LipaseB (black), Lipoprotein Lipase (grey), and Rabbit Liver Esterase (white)enzymes in protein-free samples containing 0.02% (w/v) PS20 with 0 and15% (w/v) HP-β-CD.

FIG. 4 displays the average (n=3) relative percent of PS80 determined byRP-ELSD for protein-free samples containing 0.02% (w/v) PS80 digestedusing 15 μg/mL of PPL for 5 hours at room temperature containing 0 and15% (w/v) HP-β-CD.

FIGS. 5A-5F display the average (n=3) (FIG. 5A) ≥1.4 (FIG. 5B) ≥2 (FIG.5C) ≥5 (FIG. 5D) ≥10 (FIG. 5E) ≥15 and (FIG. 5F) ≥25 μM subvisibleparticle counts per milliliter for samples digested using 15 μg/mL PPLfor 5 hours at room temperature in protein-free samples containing 0.02%(w/v) PS80 and 0 and 15% (w/v) HP-β-CD.

FIG. 6 displays the average (n=3) relative percent of PS20 determined byRP-ELSD for samples digested with 15 μg/mL of PPL enzyme at roomtemperature in protein-free samples containing 15% (w/v) of sucrose(circles), HP-α-CD (diamonds), and HP-β-CD (triangles) as a function oftime.

FIG. 7 displays the average (n=3) relative percent of PS20 determined byRP-ELSD for samples digested using 15 μg/mL of PPL enzyme for 4.5 hoursat room temperature in protein-free samples containing 0.02% (w/v) PS20with no excipient (control), 15% (w/v) sucrose, 1% (w/v) methionine, 15%(w/v) PEG 1500, 15% (w/v) PVP, 15% (w/v) HP-α-CD, 15% (w/v) HP-β-CD, 15%(w/v) SBE-β-CD, and 15% (w/v) HP-γ-CD.

FIGS. 8A-8D display the average (n=3) ≥2 μM (FIG. 8A), ≥5 μM (FIG. 8B),≥10 μM (FIG. 8C), and ≥25 μM (FIG. 8D) particle counts per milliliterdetermined by HIAC for samples digested using 15 μg/mL of PPL enzyme for4.5 hours at room temperature in protein-free samples containing 0.02%(w/v) PS20 with no excipient (control), 15% (w/v) sucrose, 1% (w/v)methionine, 15% (w/v) PEG 1500, 15% (w/v) PVP, 15% (w/v) HP-α-CD, 15%(w/v) HP-β-CD, 15% (w/v) SBE-β-CD, and 15% (w/v) HP-γ-CD.

FIG. 9 displays the average (n=3) relative percent of PS20 determined byRP-ELSD for samples digested using Candida Antarctica Lipase B inprotein-free samples containing 0.02 (w/v) PS20 with no excipient(control), 15% (w/v) SBE-β-CD, 15% (w/v) HP-α-CD, 15% (w/v) HP-β-CD, 15%(w/v) HP-γ-CD, and 15% (w/v) sucrose.

FIGS. 10A and 10B display the average (n=3) (FIG. 10A) ≥2 μM, and (FIG.10B) ≥5 μM particle counts per milliliter determined by HIAC for samplesafter addition of various excipients (HP-α-CD, HP-β-CD, HP-γ-CD,SBE-β-CD, PVP, PEG 1500, sucrose, and methionine) to evaluate there-solubilization of existing particles produced as a result ofenzymatic PS-20 degradation.

FIGS. 11A and 11B display a vial containing PS20-related particlesgenerated by enzymatic digestion using 15 μg/mL of PPL enzyme for 4.5hours at room temperature (FIG. 11A) before, and (FIG. 11B) afterspiking in 15.0% (w/v) HP-β-CD. Following addition of 15% (w/v) HP-β-CD,there are no visible particles.

FIGS. 12A-12F display the average (n=3) (FIG. 12A) ≥1.4 μM, (FIG. 12B)≥2 μM, (FIG. 12C) ≥5 μM, (FIG. 12D) ≥10 μM, (FIG. 12E) ≥15 μM, and (FIG.12F) ≥25 μM subvisible particle counts determined by HIAC per milliliterin protein-free samples containing 0.02% (w/v) PS20 stored for 27 monthsat 5° C.

FIG. 13 displays the average (n=3) relative percent of PS20 determinedby RP-ELSD for samples digested using 15 μg/mL of PPL enzyme for 4.5hours at room temperature in protein-free samples containing 0.02% (w/v)PS20 and different amounts of HP-β-CD. Data is fit using a sigmoidalmodel.

FIGS. 14A-14D display the average (n=3) relative percent of PS20determined by RP-ELSD for samples containing (FIG. 14A) 0.005%, (FIG.14B) 0.02%, (FIG. 14C) 0.1%, and (FIG. 14D) 0.4% PS20 digested using 15μg/mL of PPL enzyme for 4.5 hours at room temperature in protein-freesamples containing no excipient (control), 0, 0.5, 5, and 15% (w/v)HP-β-CD.

FIGS. 15A-15C display panel bar plot displays the average (n=3) (FIG.15A) ≥2 μM, (FIG. 15B) ≥5 μM, (FIG. 15C) ≥10 μM particle counts permilliliter determined by HIAC for samples digested using 15 μg/mL of PPLenzyme for 4.5 hours at room temperature in protein-free samplescontaining 0.02% PS20 and 0, 0.1, 0.5, 5, and 15% (w/v) HP-β-CD.

FIG. 16 displays the average (n=3) relative percent of PS20 determinedby RP-ELSD for samples digested using 15 μg/mL of PPL enzyme for 4.5hours at room temperature in protein-free samples containing differentHP-β-CD to PS20 molar ratios. Data is fit using a sigmoidal model.

FIGS. 17A-17D display the relative percent of PS20 determined by RP-ELSDfor (FIG. 17A) Control, (FIG. 17B) monoclonal antibody (mAb), (FIG. 17C)bispecific antibody (BsAb), and (FIG. 17D) single Fab antibody (sFAb)samples digested using 15 μg/mL of PPL enzyme for 4.5 hours at roomtemperature containing 0, 5, and 15% (w/v) HP-β-CD.

DETAILED DESCRIPTION

The invention herein relates to methods of reducing polysorbatedegradation in an aqueous formulation comprising a polysorbate by addinga cyclodextrin to the formulation, wherein the resulting w/w ratio ofcyclodextrin to polysorbate is greater than about 37.5:1. The inventionalso provides methods of reducing the amount of sub-visible and visibleparticles in an aqueous solution and methods of disaggregating andsolubilizing polysorbate degradation products comprising polysorbatecomprising adding cyclodextrin to a solution wherein the ratio ofcyclodextrin to polysorbate is greater than about 37.5:1. The inventionfurther provides stable aqueous formulations comprising a polysorbate,and a cyclodextrin, wherein the w/w ratio of cyclodextrin to polysorbatein the formulation is at least about 37.5:1. In some embodiments, theformulation further comprises a polypeptide.

I. Definitions

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulations are sterile.

A “sterile” formulation is aseptic or free or essentially free from allliving microorganisms and their spores.

A “stable” formulation is one in which the protein therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon storage. Preferably, the formulationessentially retains its physical and chemical stability, as well as itsbiological activity upon storage. A stable formulation also may retainits level of polysorbate upon storage. The storage period is generallyselected based on the intended shelf-life of the formulation. Variousanalytical techniques for measuring protein stability are available inthe art and are reviewed in Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) andJones, Adv. Drug Delivery Rev. 10: 29-90 (1993), for example. Stabilitycan be measured at a selected amount of light exposure and/ortemperature for a selected time period. Stability can be evaluatedqualitatively and/or quantitatively in a variety of different ways,including evaluation of aggregate formation (for example using sizeexclusion chromatography, by measuring turbidity, and/or by visualinspection); evaluation of ROS formation (for example by using a lightstress assay or a 2,2′-Azobis(2-Amidinopropane) Dihydrochloride (AAPH)stress assay); oxidation of specific amino acid residues of the protein(for example a Trp residue and/or a Met residue of a monoclonalantibody); by assessing charge heterogeneity using cation exchangechromatography, image capillary isoelectric focusing (icIEF) orcapillary zone electrophoresis; amino-terminal or carboxy-terminalsequence analysis; mass spectrometric analysis; SDS-PAGE analysis tocompare reduced and intact antibody; peptide map (for example tryptic orLYS-C) analysis; evaluating biological activity or target bindingfunction of the protein (e.g., antigen binding function of an antibody);etc. Instability may involve any one or more of: aggregation,deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation and/orTrp oxidation), isomerization (e.g. Asp isomeriation),clipping/hydrolysis/fragmentation (e.g. hinge region fragmentation),succinimide formation, unpaired cysteine(s), N-terminal extension,C-terminal processing, glycosylation differences, etc.

A protein “retains its physical stability” in a pharmaceuticalformulation if it shows no signs or very little of aggregation,precipitation and/or denaturation upon visual examination of colorand/or clarity, or as measured by UV light scattering or by sizeexclusion chromatography.

A protein “retains its chemical stability” in a pharmaceuticalformulation, if the chemical stability at a given time is such that theprotein is considered to still retain its biological activity as definedbelow. Chemical stability can be assessed by detecting and quantifyingchemically altered forms of the protein. Chemical alteration may involveprotein oxidation which can be evaluated using tryptic peptide mapping,reverse-phase high-performance liquid chromatography (HPLC) and liquidchromatography-mass spectrometry (LC/MS), for example. Other types ofchemical alteration include charge alteration of the protein which canbe evaluated by ion-exchange chromatography or icIEF, for example.

A protein “retains its biological activity” in a pharmaceuticalformulation, if the biological activity of the protein at a given timeis within about 10% (within the errors of the assay) of the biologicalactivity exhibited at the time the pharmaceutical formulation wasprepared as determined for example in an antigen binding assay for amonoclonal antibody.

As used herein, “biological activity” of a protein refers to the abilityof the protein to bind its target, for example the ability of amonoclonal antibody to bind to an antigen. It can further include abiological response which can be measured in vitro or in vivo. Suchactivity may be antagonistic or agonistic.

A protein which is “susceptible to oxidation” is one comprising one ormore residue(s) that has been found to be prone to oxidation such as,but not limited to, methionine (Met), cysteine (Cys), histidine (His),tryptophan (Trp), and tyrosine (Tyr). For example, a tryptophan aminoacid in the Fab portion of a monoclonal antibody or a methionine aminoacid in the Fc portion of a monoclonal antibody may be susceptible tooxidation.

By “isotonic” is meant that the formulation of interest has essentiallythe same osmotic pressure as human blood. Isotonic formulations willgenerally have an osmotic pressure from about 250 to 350 mOsm.Isotonicity can be measured using a vapor pressure or ice-freezing typeosmometer, for example.

As used herein, “buffer” refers to a buffered solution that resistschanges in pH by the action of its acid-base conjugate components. Thebuffer of this invention preferably has a pH in the range from about 4.5to about 8.0. For example, histidine acetate is an example of a bufferthat will control the pH in this range.

A “preservative” is a compound which can be optionally included in theformulation to essentially reduce bacterial action therein, thusfacilitating the production of a multi-use formulation, for example.Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (amixture of alkylbenzyldimethylammonium chlorides in which the alkylgroups are long-chain compounds), and benzethonium chloride. Other typesof preservatives include aromatic alcohols such as phenol, butyl andbenzyl alcohol, alkyl parabens such as methyl or propyl paraben,catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. In oneembodiment, the preservative herein is benzyl alcohol.

As used herein, a “surfactant” refers to a surface-active agent,preferably a nonionic surfactant. Examples of surfactants herein includepolysorbate (for example, polysorbate 20 and, polysorbate 80); poloxamer(e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodiumlaurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-,or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT′ series (Mona Industries, Inc.,Paterson, N.J.); polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc); etc.

“Pharmaceutically acceptable” excipients or carriers as used hereininclude pharmaceutically acceptable carriers, stabilizers, buffers,acids, bases, sugars, preservatives, surfactants, tonicity agents, andthe like, which are well known in the art (Remington: The Science andPractice of Pharmacy, 22nd Ed., Pharmaceutical Press, 2012). Examples ofpharmaceutically acceptable excipients include buffers such asphosphate, citrate, acetate, and other organic acids; antioxidantsincluding ascorbic acid, L-tryptophan and methionine; low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone (PVP); amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; metalcomplexes such as Zn-protein complexes; chelating agents such as EDTA;sugar alcohols such as mannitol or sorbitol; salt-forming counterionssuch as sodium; and/or nonionic surfactants such as polysorbate,poloxamer, polyethylene glycol (PEG), and PLURONICS™. “Pharmaceuticallyacceptable” excipients or carriers are those which can reasonably beadministered to a subject to provide an effective dose of the activeingredient employed and that are nontoxic to the subject being exposedthereto at the dosages and concentrations employed.

The term “polysorbate” (also abbreviated as PS) as used herein refers toPEGylated sorbitan esterified with fatty acids and includes polysorbate20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40(polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60(polyoxyethylene (20) sorbitan monostearate), and polysorbate 80(polyoxyethylene (20) sorbitan monooleate).

The term “cyclodextrin” refers to a family of compounds comprisingglucose molecules bound in a ring-like structure with d-glucopyranoseunits linked with alpha-(1,4) glycosidic bonds. Exemplary cyclodextrinsinclude 2-hydroxypropyl-β-cyclodextrin (HP-β-CD orHP-beta-cyclodextrin), 2-hydroxypropyl-α-cyclodextrin (HP-α-CD orHP-alpha-cyclodextrin), 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD orHP-gamma-cyclodextrin), β-cyclodextrin (β-CD or beta-cyclodextrin),sulfobutyl ether β-cyclodextrin (SBE-β-CD or SBE-beta-cyclodextrin),α-cyclodextrin (α-CD or alpha-cyclodextrin), and γ-cyclodextrin (γ-CD orgamma-cyclodextrin). Synonyms for cyclodextrin include Cavitron, cyclicoligosaccharide, cycloamulose, and cycloglucan.

The term “tonicity agent” refers to an agent that is used to adjust ormaintain the relative concentration of solutions. Preferred tonicityagents include polyhydric sugar alcohols, preferably trihydric or highersugar alcohols, such as glycerin, erythritol, arabitol, xylitol,sorbitol and mannitol.

The term “stabilizer” refers to agents that stabilize large chargedbiomolecules, such as proteins and antibodies. Tonicity agents may alsoserve as stabilizers, when used with large charged biomolecules.

“Reduced polysorbate degradation” refers to conditions, where after aperiod of time more polysorbate remains in a sample compared to acontrol under similar storage conditions. For example, a sample that has95% polysorbate remaining after a period of time shows reducedpolysorbate degradation compared to a control sample that has 50% ofpolysorbate remaining after the same time period.

The term “disaggregate” as used herein refers to the reduction invisible and/or subvisible particles that are caused by polysorbatedegradation. For example, an agent is effective at disaggregatingpolysorbate degradation products if the amounts of visible and/orsubvisible particles are reduced when it is added to a solutioncontaining polysorbate.

The term “solubilize” refers to dissolving a solid in a liquid. Forexample, an agent is effective at solubilizing a compound, if thecompound dissolves more readily in that agent's presence.

The term “w/w ratio” refers to the amount of one solute by mass dividedby the amount of another solute by mass. For example, a solution thatcontains 100 mg of cyclodextrin and 1 mg of polysorbate, has a w/w ratioof cyclodextrin to polysorbate of 100:1. According to one embodiment thew/w ratio of cyclodextrin to polysorbate is greater than about 37.5:1.

An “aqueous formulation” refers to a water-based liquid formulationsuitable for administration. The formulation may contain a therapeuticagent, such as an antibody or small molecules and is preferably sterile.Aqueous formulations may also contain buffers, stabilizers, tonicityagents, and excipients.

The protein which is formulated is preferably essentially pure anddesirably essentially homogeneous (e.g., free from contaminatingproteins etc.). “Essentially pure” protein means a compositioncomprising at least about 90% by weight of the protein (e.g., monoclonalantibody), based on total weight of the composition, preferably at leastabout 95% by weight. “Essentially homogeneous” protein means acomposition comprising at least about 99% by weight of the protein(e.g., monoclonal antibody), based on total weight of the composition.

The terms “protein” “polypeptide” and “peptide” are used interchangeablyherein to refer to polymers of amino acids of any length. The polymermay be linear or branched, it may comprise modified amino acids, and itmay be interrupted by non-amino acids. The terms also encompass an aminoacid polymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, proteins containing one or more analogs ofan amino acid (including, for example, unnatural amino acids, etc.), aswell as other modifications known in the art. Examples of proteinsencompassed within the definition herein include mammalian proteins,such as, e.g., renin; a growth hormone, including human growth hormoneand bovine growth hormone; growth hormone releasing factor; parathyroidhormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;insulin A-chain; insulin B-chain; proinsulin; follicle stimulatinghormone; calcitonin; luteinizing hormone; glucagon; leptin; clottingfactors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; a tumor necrosis factor receptor such as deathreceptor 5 and CD120; TNF-related apoptosis-inducing ligand (TRAIL);B-cell maturation antigen (BCMA); B-lymphocyte stimulator (BLyS); aproliferation-inducing ligand (APRIL); enkephalinase; RANTES (regulatedon activation normally T-cell expressed and secreted); human macrophageinflammatory protein (MIP-1-alpha); a serum albumin such as human serumalbumin; Muellerian-inhibiting substance; relaxin A-chain; relaxinB-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbialprotein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyteassociated antigen (CTLA), such as CTLA-4; inhibin; activin;platelet-derived endothelial cell growth factor (PD-ECGF); a vascularendothelial growth factor family protein (e.g., VEGF-A, VEGF-B, VEGF-C,VEGF-D, and P1GF); a platelet-derived growth factor (PDGF) familyprotein (e.g., PDGF-A, PDGF-B, PDGF-C, PDGF-D, and dimers thereof);fibroblast growth factor (FGF) family such as aFGF, bFGF, FGF4, andFGF9; epidermal growth factor (EGF); receptors for hormones or growthfactors such as a VEGF receptor(s) (e.g., VEGFR1, VEGFR2, and VEGFR3),epidermal growth factor (EGF) receptor(s) (e.g., ErbB1, ErbB2, ErbB3,and ErbB4 receptor), platelet-derived growth factor (PDGF) receptor(s)(e.g., PDGFR-α and PDGFR-β), and fibroblast growth factor receptor(s);TIE ligands (Angiopoietins, ANGPT1, ANGPT2); Angiopoietin receptor suchas TIE1 and TIE2; protein A or D; rheumatoid factors; a neurotrophicfactor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3,-4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor suchas NGF-b; transforming growth factor (TGF) such as TGF-alpha andTGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5;insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I(brain IGF-I), insulin-like growth factor binding proteins (IGFBPs); CDproteins such as CD3, CD4, CD8, CD19 and CD20; erythropoietin;osteoinductive factors; immunotoxins; a bone morphogenetic protein(BMP); a chemokine such as CXCL12 and CXCR4; an interferon such asinterferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs),e.g., M-CSF, GM-CSF, and G-CSF; a cytokine such as interleukins (ILs),e.g., IL-1 to IL-10; midkine; superoxide dismutase; T-cell receptors;surface membrane proteins; decay accelerating factor; viral antigen suchas, for example, a portion of the AIDS envelope; transport proteins;homing receptors; addressins; regulatory proteins; integrins such asCD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM; ephrins; Bv8;Delta-like ligand 4 (DLL4); Del-1; BMP9; BMP10; Follistatin; Hepatocytegrowth factor (HGF)/scatter factor (SF); Alk1; Robo4; ESM1; Perlecan;EGF-like domain, multiple 7 (EGFL7); CTGF and members of its family;thrombospondins such as thrombospondinl and thrombospondin2; collagenssuch as collagen IV and collagen XVIII; neuropilins such as NRP1 andNRP2; Pleiotrophin (PTN); Progranulin; Proliferin; Notch proteins suchas Notchl and Notch4; semaphorins such as Sema3A, Sema3C, and Sema3F; atumor associated antigen such as CA125 (ovarian cancer antigen);immunoadhesins; and fragments and/or variants of any of the above-listedproteins as well as antibodies, including antibody fragments, binding toone or more protein, including, for example, any of the above-listedproteins.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments so long as theyexhibit the desired biological activity.

An “isolated” protein (e.g., an isolated antibody) is one which has beenidentified and separated and/or recovered from a component of itsnatural environment. Contaminant components of its natural environmentare materials which would interfere with research, diagnostic ortherapeutic uses for the protein, and may include enzymes, hormones, andother proteinaceous or nonproteinaceous solutes. Isolated proteinincludes the protein in situ within recombinant cells since at least onecomponent of the protein's natural environment will not be present.Ordinarily, however, isolated protein will be prepared by at least onepurification step.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The term “constant domain” refers to the portion of an immunoglobulinmolecule having a more conserved amino acid sequence relative to theother portion of the immunoglobulin, the variable domain, which containsthe antigen binding site. The constant domain contains the CH1, CH2 andCH3 domains (collectively, CH) of the heavy chain and the CHL (or CL)domain of the light chain.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH.” Thevariable domain of the light chain may be referred to as “VL.” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions (HVRs) or both in thelight-chain and the heavy-chain variable domains. In some embodiments,the HVRs are Complementarity Determining Regions (CDRs).

The more highly conserved portions of variable domains are called theframework regions (FR). The variable domains of native heavy and lightchains each comprise four FR regions, largely adopting a beta-sheetconfiguration, connected by three HVRs, which form loops connecting, andin some cases forming part of, the beta-sheet structure. The HVRs ineach chain are held together in close proximity by the FR regions and,with the HVRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. (1991)). The constant domains are not involveddirectly in the binding of an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any mammalianspecies can be assigned to one of two clearly distinct types, calledkappa (“κ”) and lambda (“λ”), based on the amino acid sequences of theirconstant domains.

The term IgG “isotype” or “subclass” as used herein is meant any of thesubclasses of immunoglobulins defined by the chemical and antigeniccharacteristics of their constant regions. Depending on the amino acidsequences of the constant domains of their heavy chains, antibodies(immunoglobulins) can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known and described generally in, for example, Abbas et al.Cellular and Mol. Immunology, 4th ed., W.B. Saunders, Co. (2000). Anantibody may be part of a larger fusion molecule, formed by covalent ornon-covalent association of the antibody with one or more other proteinsor peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containan Fc region.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen. The Fab fragment contains the heavy- and light-chain variabledomains and also contains the constant domain of the light chain and thefirst constant domain (CH1) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxyterminus of the heavy chain CH1 domain including one or more cysteinesfrom the antibody hinge region. Fab′-SH is the designation herein forFab′ in which the cysteine residue(s) of the constant domains bear afree thiol group. F(ab′)₂ antibody fragments originally were produced aspairs of Fab′ fragments which have hinge cysteines between them. Otherchemical couplings of antibody fragments are also known.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three HVRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six HVRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFv,see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); andHollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., Nat.Med. 9:129-134 (2003).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,e.g., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the invention may be made by avariety of techniques, including, for example, the hybridoma method(e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al.,Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas pp.563-681 Elsevier, N.Y. (1981)), recombinant DNA methods (see, e.g., U.S.Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson etal., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Leeet al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl.Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol.Methods 284(1-2): 119-132 (2004), and technologies for producing humanor human-like antibodies in animals that have parts or all of the humanimmunoglobulin loci or genes encoding human immunoglobulin sequences(see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741;Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993);Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Yearin Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;5,625,126; 5,633,425; and U.S. Pat. No. 5,661,016; Marks et al.,Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859(1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., NatureBiotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826(1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995)).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (see, e.g., U.S. Pat. No. 4,816,567; andMorrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Chimeric antibodies include PRIMATIZED® antibodies wherein theantigen-binding region of the antibody is derived from an antibodyproduced by, e.g., immunizing macaque monkeys with the antigen ofinterest.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from a HVR of therecipient are replaced by residues from a HVR of a non-human species(donor antibody) such as mouse, rat, rabbit, or nonhuman primate havingthe desired specificity, affinity, and/or capacity. In some instances,FR residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, e.g., Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g.,Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the HVR residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody.

The expression “linear antibodies” refers to the antibodies described inZapata et al. (Protein Eng., 8(10):1057-1062 (1995)). Briefly, theseantibodies comprise a pair of tandem Fc segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen (especially when synthetic peptides are used) to a protein thatis immunogenic in the species to be immunized. For example, the antigencan be conjugated to keyhole limpet hemocyanin (KLH), serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctionalor derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

The term “multispecific antibody” is used in the broadest sense andspecifically covers an antibody comprising an antigen-binding domainthat has polyepitopic specificity (i.e., is capable of specificallybinding to two, or more, different epitopes on one biological moleculeor is capable of specifically binding to epitopes on two, or more,different biological molecules). In some embodiments, an antigen-bindingdomain of a multispecific antibody (such as a bispecific antibody)comprises two VH/VL units, wherein a first VH/VL unit specifically bindsto a first epitope and a second VH/VL unit specifically binds to asecond epitope, wherein each VH/VL unit comprises a heavy chain variabledomain (VH) and a light chain variable domain (VL). Such multispecificantibodies include, but are not limited to, full length antibodies,antibodies having two or more VL and VH domains, antibody fragments suchas Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies,antibody fragments that have been linked covalently or non-covalently. AVH/VL unit that further comprises at least a portion of a heavy chainconstant region and/or at least a portion of a light chain constantregion may also be referred to as a “hemimer” or “half antibody.” Insome embodiments, a half antibody comprises at least a portion of asingle heavy chain variable region and at least a portion of a singlelight chain variable region. In some such embodiments, a bispecificantibody that comprises two half antibodies and binds to two antigenscomprises a first half antibody that binds to the first antigen or firstepitope but not to the second antigen or second epitope and a secondhalf antibody that binds to the second antigen or second epitope and notto the first antigen or first epitope. According to some embodiments,the multispecific antibody is an IgG antibody that binds to each antigenor epitope with an affinity of 5 M to 0.001 pM, 3 M to 0.001 pM, 1 M to0.001 pM, 0.5 M to 0.001 pM, or 0.1 M to 0.001 pM. In some embodiments,a hemimer comprises a sufficient portion of a heavy chain variableregion to allow intramolecular disulfide bonds to be formed with asecond hemimer. In some embodiments, a hemimer comprises a knob mutationor a hole mutation, for example, to allow heterodimerization with asecond hemimer or half antibody that comprises a complementary holemutation or knob mutation. Knob mutations and hole mutations arediscussed further below.

A “bispecific antibody” is a multispecific antibody comprising anantigen-binding domain that is capable of specifically binding to twodifferent epitopes on one biological molecule or is capable ofspecifically binding to epitopes on two different biological molecules.A bispecific antibody may also be referred to herein as having “dualspecificity” or as being “dual specific.” Unless otherwise indicated,the order in which the antigens bound by a bispecific antibody arelisted in a bispecific antibody name is arbitrary. In some embodiments,a bispecific antibody comprises two half antibodies, wherein each halfantibody comprises a single heavy chain variable region and optionallyat least a portion of a heavy chain constant region, and a single lightchain variable region and optionally at least a portion of a light chainconstant region. In certain embodiments, a bispecific antibody comprisestwo half antibodies, wherein each half antibody comprises a single heavychain variable region and a single light chain variable region and doesnot comprise more than one single heavy chain variable region and doesnot comprise more than one single light chain variable region. In someembodiments, a bispecific antibody comprises two half antibodies,wherein each half antibody comprises a single heavy chain variableregion and a single light chain variable region, and wherein the firsthalf antibody binds to a first antigen and not to a second antigen andthe second half antibody binds to the second antigen and not to thefirst antigen.

The term “knob-into-hole” or “KnH” technology as used herein refers tothe technology directing the pairing of two polypeptides together invitro or in vivo by introducing a protuberance (knob) into onepolypeptide and a cavity (hole) into the other polypeptide at aninterface in which they interact. For example, KnHs have been introducedin the Fc:Fc binding interfaces, CL:CH1 interfaces or VH/VL interfacesof antibodies (see, e.g., US 2011/0287009, US2007/0178552, WO 96/027011,WO 98/050431, and Zhu et al., 1997, Protein Science 6:781-788). In someembodiments, KnHs drive the pairing of two different heavy chainstogether during the manufacture of multispecific antibodies. Forexample, multispecific antibodies having KnH in their Fc regions canfurther comprise single variable domains linked to each Fc region, orfurther comprise different heavy chain variable domains that pair withsimilar or different light chain variable domains. KnH technology canalso be used to pair two different receptor extracellular domainstogether or any other polypeptide sequences that comprises differenttarget recognition sequences (e.g., including affibodies, peptibodiesand other Fc fusions).

The term “knob mutation” as used herein refers to a mutation thatintroduces a protuberance (knob) into a polypeptide at an interface inwhich the polypeptide interacts with another polypeptide. In someembodiments, the other polypeptide has a hole mutation (see e.g., U.S.Pat. Nos. 5,731,168, 5,807,706, 5,821,333, 7,695,936, 8,216,805, eachincorporated herein by reference in its entirety).

The term “hole mutation” as used herein refers to a mutation thatintroduces a cavity (hole) into a polypeptide at an interface in whichthe polypeptide interacts with another polypeptide. In some embodiments,the other polypeptide has a knob mutation (see e.g., U.S. Pat. Nos.5,731,168, 5,807,706, 5,821,333, 7,695,936, 8,216,805, each incorporatedherein by reference in its entirety).

The term “about” as used herein refers to an acceptable error range forthe respective value as determined by one of ordinary skill in the art,which will depend in part how the value is measured or determined, i.e.,the limitations of the measurement system. For example, “about” can meanwithin 1 or more than 1 standard deviations, per the practice in theart. A reference to “about” a value or parameter herein includes anddescribes embodiments that are directed to that value or parameter perse. For example, a description referring to “about X” includesdescription of “X”.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a compound”optionally includes a combination of two or more such compounds, and thelike.

II. Formulations and Preparation

The invention herein relates to methods of reducing polysorbatedegradation in an aqueous formulation comprising polysorbate, the methodcomprising adding a cyclodextrin to the formulation, wherein theresulting w/w ratio of cyclodextrin to polysorbate is greater than about37.5:1. In some embodiments, the invention provides a method of reducingthe amount of sub-visible and visible particles in an aqueous solutioncomprising polysorbate comprising adding cyclodextrin to a solutionwherein the resulting w/w ratio of cyclodextrin to polysorbate isgreater than about 37.5:1. In some embodiments, the invention provides amethod to disaggregate and solubilize polysorbate degradation productsin an aqueous formulation comprising adding a cyclodextrin to theformulation, wherein the resulting w/w ratio of cyclodextrin topolysorbate is greater than about 37.5:1. In some embodiments, thecyclodextrin is HP-βcyclodextrin, HP-γcyclodextrin or sulfobutyl etherbeta-cyclodextrin. In some embodiments, the cyclodextrin is HP-αcyclodextrin. In some embodiments, the formulation further comprises apolypeptide.

In some embodiments, the method comprises adding polyvinylpyrrolidone(PVP) to the formulation, wherein the resulting w/w ratio of PVP topolysorbate is greater than about 37.5:1. In some embodiments, theinvention provides a method of reducing the amount of sub-visible andvisible particles in an aqueous solution comprising polysorbatecomprising adding PVP to a solution wherein resulting w/w ratio of PVPto polysorbate is greater than about 37.5:1. In some embodiments, theinvention provides a method to disaggregate and solubilize polysorbatedegradation products in an aqueous formulation comprising adding PVP tothe formulation, wherein the resulting w/w ratio of PVP to polysorbateis greater than about 37.5:1.

In some embodiments, the invention provides an aqueous formulationcomprising a polysorbate, and a cyclodextrin, wherein less than 1% ofthe polysorbate has been degraded after storage at about 1° C. to about10° C. for at least about six months to at least about 48 months,wherein the w/w ratio of cyclodextrin to polysorbate in the formulationis at least about 37.5:1. In some embodiments, the invention provides anaqueous formulation comprising a polysorbate and PVP, wherein less than1% of the polysorbate has been degraded after storage at about 1° C. toabout 10° C. for at least about six months to at least about 48 months,wherein the w/w ratio of PVP to polysorbate in the formulation is atleast about 37.5:1. In some embodiments, the formulation is stable atabout 2° C. to about 8° C. for at least about six months to at leastabout at least about 48 months, at least about 12 months, at least about18 months, at least about 24 months, or at least about 48 months. Insome embodiments, the formulation comprises about 0.005%-0.4%polysorbate. In some embodiments, the formulation comprises about0.005%-0.4% polysorbate and the cyclodextrin is added to the formulationto a concentration of about 0.01%-30%. In some embodiments, thecyclodextrin is HP-βcyclodextrin, HP-γcyclodextrin or sulfobutyl etherbeta-cyclodextrin. In some embodiments, the cyclodextrin is HP-αcyclodextrin. In some embodiments the polysorbate degradation is reducedby about 50%, about 75%, about 80%, about 85%, about 90%, about 95%, orabout 99%. In further embodiments, less than about 1,000, about 750,about 500, about 250, about 150, about 100, about 50, or about 25polysorbate particles greater than about two microns in diameter areformed per mL. In some embodiments, the formulation further comprises apolypeptide. In some embodiments, the protein concentration is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein concentrationis greater than about 250 mg/mL. In some embodiments, the formulationhas a pH of about 4.5 to about 7.0 or about 4.5 to about 6.0, or ofabout 6.0. In some embodiments, the formulation further comprises one ormore of a stabilizer, a buffer, a surfactant, and a tonicity agent. Infurther embodiments, the formulation is suitable for intravenous,subcutaneous intramuscular, or intravitreal administration to a subject.In some embodiments, the polypeptide is a polyclonal antibody, amonoclonal antibody, a humanized antibody, a human antibody, a chimericantibody, a multispecific antibody, or an antibody fragment. In someembodiments, the formulation further comprises a small molecule, anucleic acid, a lipid and/or a carbohydrate.

Proteins and antibodies in the formulation may be prepared using methodsknown in the art. Provided herein are non-limiting exemplary methods forpreparing an antibody (e.g., full length antibodies, antibody fragmentsand multispecific antibodies). The antibody in the aqueous formulationis prepared using techniques available in the art for generatingantibodies, exemplary methods of which are described in more detail inthe following sections. The methods herein can be adapted by one ofskill in the art for the preparation of formulations comprising otherproteins such as peptide-based inhibitors. See Sam Sambrook et al.,Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2012); Current Protocols inMolecular Biology, F. M. Ausubel, et al. eds. (2003); Short Protocols inMolecular Biology, Ausubel et al., eds., J. Wiley and Sons (2002);Horswill et al., Current Protocols in Protein Science, (2006);Antibodies, A Laboratory Manual, Harlow and Lane, eds. (1988); R. I.Freshney, Culture of Animal Cells: A Manual of Basic Technique andSpecialized Application, 6th ed., J. Wiley and Sons (2010) for generallywell understood and commonly employed techniques and procedures for theproduction of therapeutic proteins, which are all incorporated herein byreference in their entirety.

A. Antibody Preparation

The antibody in the aqueous formulations provided herein is directedagainst an antigen of interest. Preferably, the antigen is abiologically important polypeptide and administration of the antibody toa mammal suffering from a disorder can result in a therapeutic benefitin that mammal. However, antibodies directed against nonpolypeptideantigens are also contemplated.

Where the antigen is a polypeptide, it may be a transmembrane molecule(e.g. receptor) or ligand such as a growth factor. Exemplary antigensinclude molecules such as vascular endothelial growth factor (VEGF);CD20; ox-LDL; ox-ApoB100; renin; a growth hormone, including humangrowth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; receptors for hormonesor growth factors; protein A or D; rheumatoid factors; a neurotrophicfactor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3,-4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor suchas NGF-β; platelet-derived growth factor (PDGF); fibroblast growthfactor such as aFGF and bFGF; epidermal growth factor (EGF);transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des (1-3)-IGF-I (brain IGF-I),insulin-like growth factor binding proteins; CD proteins such as CD3,CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors;immunotoxins; a bone morphogenetic protein (BMP); an interferon such asinterferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs),e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;superoxide dismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of any of the above-listed polypeptides.

(i) Antigen Preparation

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g. theextracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Otherantigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

(ii) Certain Antibody-Based Methods

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

Monoclonal antibodies of the invention can be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), andfurther described, e.g., in Hongo et al., Hybridoma, 14 (3): 253-260(1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) regardinghuman-human hybridomas. Additional methods include those described, forexample, in U.S. Pat. No. 7,189,826 regarding production of monoclonalhuman natural IgM antibodies from hybridoma cell lines. Human hybridomatechnology (Trioma technology) is described in Vollmers and Brandlein,Histology and Histopathology, 20(3):927-937 (2005) and Vollmers andBrandlein, Methods and Findings in Experimental and ClinicalPharmacology, 27(3):185-91 (2005).

For various other hybridoma techniques, see, e.g., US 2006/258841; US2006/183887 (fully human antibodies), US 2006/059575; US 2005/287149; US2005/100546; US 2005/026229; and U.S. Pat. Nos. 7,078,492 and 7,153,507.An exemplary protocol for producing monoclonal antibodies using thehybridoma method is described as follows. In one embodiment, a mouse orother appropriate host animal, such as a hamster, is immunized to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization. Antibodiesare raised in animals by multiple subcutaneous (sc) or intraperitoneal(ip) injections of a polypeptide of the invention or a fragment thereof,and an adjuvant, such as monophosphoryl lipid A (MPL)/trehalosedicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton,Mont.). A polypeptide of the invention (e.g., antigen) or a fragmentthereof may be prepared using methods well known in the art, such asrecombinant methods, some of which are further described herein. Serumfrom immunized animals is assayed for anti-antigen antibodies, andbooster immunizations are optionally administered. Lymphocytes fromanimals producing anti-antigen antibodies are isolated. Alternatively,lymphocytes may be immunized in vitro.

Lymphocytes are then fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell. See, e.g.,Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103Academic Press, (1986). Myeloma cells may be used that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Exemplary myeloma cells include, but are not limited to, murinemyeloma lines, such as those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from theAmerican Type Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 Marcel Dekker, Inc., New York (1987).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium, e.g., a medium that contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells. Preferably, serum-free hybridoma cell culturemethods are used to reduce use of animal-derived serum such as fetalbovine serum, as described, for example, in Even et al., Trends inBiotechnology, 24(3), 105-108 (2006).

Oligopeptides as tools for improving productivity of hybridoma cellcultures are described in Franek, Trends in Monoclonal AntibodyResearch, 111-122 (2005). Specifically, standard culture media areenriched with certain amino acids (alanine, serine, asparagine,proline), or with protein hydrolyzate fractions, and apoptosis may besignificantly suppressed by synthetic oligopeptides, constituted ofthree to six amino acid residues. The peptides are present at millimolaror higher concentrations.

Culture medium in which hybridoma cells are growing may be assayed forproduction of monoclonal antibodies that bind to an antibody of theinvention. The binding specificity of monoclonal antibodies produced byhybridoma cells may be determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (MA) or enzyme-linkedimmunoadsorbent assay (ELISA). The binding affinity of the monoclonalantibody can be determined, for example, by Scatchard analysis. See,e.g., Munson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.See, e.g., Goding, supra. Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, hybridomacells may be grown in vivo as ascites tumors in an animal. Monoclonalantibodies secreted by the subclones are suitably separated from theculture medium, ascites fluid, or serum by conventional immunoglobulinpurification procedures such as, for example, protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography. One procedure for isolation of proteins fromhybridoma cells is described in US 2005/176122 and U.S. Pat. No.6,919,436. The method includes using minimal salts, such as lyotropicsalts, in the binding process and preferably also using small amounts oforganic solvents in the elution process.

(iii) Certain Library Screening Methods

Antibodies of the invention can be made by using combinatorial librariesto screen for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are describedgenerally in Hoogenboom et al. in Methods in Molecular Biology 178:1-37,O'Brien et al., ed., Human Press, Totowa, N.J., (2001). For example, onemethod of generating antibodies of interest is through the use of aphage antibody library as described in Lee et al., J. Mol. Biol.340(5):1073-93 (2004).

In principle, synthetic antibody clones are selected by screening phagelibraries containing phage that display various fragments of antibodyvariable region (Fv) fused to phage coat protein. Such phage librariesare panned by affinity chromatography against the desired antigen.Clones expressing Fv fragments capable of binding to the desired antigenare adsorbed to the antigen and thus separated from the non-bindingclones in the library. The binding clones are then eluted from theantigen, and can be further enriched by additional cycles of antigenadsorption/elution. Any of the antibodies of the invention can beobtained by designing a suitable antigen screening procedure to selectfor the phage clone of interest followed by construction of a fulllength antibody clone using the Fv sequences from the phage clone ofinterest and suitable constant region (Fc) sequences described in Kabatet al., Sequences of Proteins of Immunological Interest, Fifth Edition,NIH Publication 1-3:91-3242, Bethesda Md. (1991).

In certain embodiments, the antigen-binding domain of an antibody isformed from two variable (V) regions of about 110 amino acids, one eachfrom the light (VL) and heavy (VH) chains, that both present threehypervariable loops (HVRs) or complementarity-determining regions(CDRs). Variable domains can be displayed functionally on phage, eitheras single-chain Fv (scFv) fragments, in which VH and VL are covalentlylinked through a short, flexible peptide, or as Fab fragments, in whichthey are each fused to a constant domain and interact non-covalently, asdescribed in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Asused herein, scFv encoding phage clones and Fab encoding phage clonesare collectively referred to as “Fv phage clones” or “Fv clones.”

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

In certain embodiments, filamentous phage is used to display antibodyfragments by fusion to the minor coat protein pIII. The antibodyfragments can be displayed as single chain Fv fragments, in which VH andVL domains are connected on the same polypeptide chain by a flexiblepolypeptide spacer, e.g. as described by Marks et al., J Mol. Biol.,222: 581-597 (1991), or as Fab fragments, in which one chain is fused topIII and the other is secreted into the bacterial host cell periplasmwhere assembly of a Fab-coat protein structure which becomes displayedon the phage surface by displacing some of the wild type coat proteins,e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137(1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-antigen clones is desired, the subject is immunizedwith antigen to generate an antibody response, and spleen cells and/orcirculating B cells other peripheral blood lymphocytes (PBLs) arerecovered for library construction. In one embodiment, a human antibodygene fragment library biased in favor of anti-antigen clones is obtainedby generating an anti-antigen antibody response in transgenic micecarrying a functional human immunoglobulin gene array (and lacking afunctional endogenous antibody production system) such that antigenimmunization gives rise to B cells producing human antibodies againstantigen. The generation of human antibody-producing transgenic mice isdescribed below.

Additional enrichment for anti-antigen reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing antigen-specific membrane bound antibody, e.g., by cellseparation using antigen affinity chromatography or adsorption of cellsto fluorochrome-labeled antigen followed by flow-activated cell sorting(FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which antigenis not antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). In certain embodiments, library diversity is maximized by usingPCR primers targeted to each V-gene family in order to amplify allavailable VH and VL arrangements present in the immune cell nucleic acidsample, e.g. as described in the method of Marks et al., J. Mol. Biol.,222: 581-597 (1991) or as described in the method of Orum et al.,Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the amplifiedDNA into expression vectors, rare restriction sites can be introducedwithin the PCR primer as a tag at one end as described in Orlandi et al.(1989), or by further PCR amplification with a tagged primer asdescribed in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 10¹² clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (K_(d) ⁻¹ of about 10⁻⁸M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique 1:11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 9607754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities of about 10⁻⁹M or less.

Screening of the libraries can be accomplished by various techniquesknown in the art. For example, antigen can be used to coat the wells ofadsorption plates, expressed on host cells affixed to adsorption platesor used in cell sorting, or conjugated to biotin for capture withstreptavidin-coated beads, or used in any other method for panning phagedisplay libraries.

The phage library samples are contacted with immobilized antigen underconditions suitable for binding at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1,000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for antigen.However, random mutation of a selected antibody (e.g. as performed insome affinity maturation techniques) is likely to give rise to manymutants, most binding to antigen, and a few with higher affinity. Withlimiting antigen, rare high affinity phage could be competed out. Toretain all higher affinity mutants, phages can be incubated with excessbiotinylated antigen, but with the biotinylated antigen at aconcentration of lower molarity than the target molar affinity constantfor antigen. The high affinity-binding phages can then be captured bystreptavidin-coated paramagnetic beads. Such “equilibrium capture”allows the antibodies to be selected according to their affinities ofbinding, with sensitivity that permits isolation of mutant clones withas little as two-fold higher affinity from a great excess of phages withlower affinity. Conditions used in washing phages bound to a solid phasecan also be manipulated to discriminate on the basis of dissociationkinetics.

Anti-antigen clones may be selected based on activity. In certainembodiments, the invention provides anti-antigen antibodies that bind toliving cells that naturally express antigen or bind to free floatingantigen or antigen attached to other cellular structures. Fv clonescorresponding to such anti-antigen antibodies can be selected by (1)isolating anti-antigen clones from a phage library as described above,and optionally amplifying the isolated population of phage clones bygrowing up the population in a suitable bacterial host; (2) selectingantigen and a second protein against which blocking and non-blockingactivity, respectively, is desired; (3) adsorbing the anti-antigen phageclones to immobilized antigen; (4) using an excess of the second proteinto elute any undesired clones that recognize antigen-bindingdeterminants which overlap or are shared with the binding determinantsof the second protein; and (5) eluting the clones which remain adsorbedfollowing step (4). Optionally, clones with the desiredblocking/non-blocking properties can be further enriched by repeatingthe selection procedures described herein one or more times.

DNA encoding hybridoma-derived monoclonal antibodies or phage display Fvclones of the invention is readily isolated and sequenced usingconventional procedures (e.g. by using oligonucleotide primers designedto specifically amplify the heavy and light chain coding regions ofinterest from hybridoma or phage DNA template). Once isolated, the DNAcan be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of the desiredmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of antibody-encoding DNA includeSkerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun,Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g. the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. An Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid,” fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In certain embodiments,an Fv clone derived from human variable DNA is fused to human constantregion DNA to form coding sequence(s) for full- or partial-length humanheavy and/or light chains.

DNA encoding anti-antigen antibody derived from a hybridoma of theinvention can also be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofhomologous murine sequences derived from the hybridoma clone (e.g. as inthe method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855(1984)). DNA encoding a hybridoma- or Fv clone-derived antibody orfragment can be further modified by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In this manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of the Fvclone or hybridoma clone-derived antibodies of the invention.

(iv) Humanized and Human Antibodies

Various methods for humanizing non-human antibodies are known in theart. For example, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one embodiment of the method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Human antibodies of the invention can be constructed by combining Fvclone variable domain sequence(s) selected from human-derived phagedisplay libraries with known human constant domain sequence(s) asdescribed above. Alternatively, human monoclonal antibodies of theinvention can be made by the hybridoma method. Human myeloma andmouse-human heteromyeloma cell lines for the production of humanmonoclonal antibodies have been described, for example, by Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, Marcel Dekker, Inc., New York,pp. 51-63 (1987); and Boerner et al., J. Immunol., 147: 86 (1991).

It is possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JO gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993);and Duchosal et al. Nature 355:258 (1992).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described herein isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

(v) Antibody Fragments

Antibody fragments may be generated by traditional means, such asenzymatic digestion, or by recombinant techniques. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors. For areview of certain antibody fragments, see Hudson et al. Nat. Med.9:129-134 (2003).

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)2 fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′) 2 fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)2 fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, an antibody is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and scFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they may be suitable forreduced nonspecific binding during in vivo use. scFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an scFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example.Such linear antibodies may be monospecific or bispecific.

(vi) Multispecific Antibodies

Multispecific antibodies have binding specificities for at least twodifferent epitopes, where the epitopes are usually from differentantigens. While such molecules normally will only bind two differentepitopes (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)2 bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO1, 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is typical to have thefirst heavy-chain constant region (CH1) containing the site necessaryfor light chain binding, present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. One interface comprises at least a part of the C_(H) 3 domainof an antibody constant domain. In this method, one or more small aminoacid side chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)2 fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)2molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al, J. Immunol, 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tuft et al., J. Immunol. 147: 60(1991).

(vii) Single-Domain Antibodies

In some embodiments, an antibody of the invention is a single-domainantibody. A single-domain antibody is a single polypeptide chaincomprising all or a portion of the heavy chain variable domain or all ora portion of the light chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody(Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516). Inone embodiment, a single-domain antibody consists of all or a portion ofthe heavy chain variable domain of an antibody.

(viii) Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodymay be prepared by introducing appropriate changes into the nucleotidesequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid alterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

(ix) Antibody Derivatives

The antibodies of the invention can be further modified to containadditional nonproteinaceous moieties that are known in the art andreadily available. In certain embodiments, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

(x) Vectors, Host Cells, Recombinant Methods

Antibodies may also be produced using recombinant methods. Forrecombinant production of an anti-antigen antibody, nucleic acidencoding the antibody is isolated and inserted into a replicable vectorfor further cloning (amplification of the DNA) or for expression. DNAencoding the antibody may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(a) Signal Sequence Component

An antibody of the invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (e.g., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process a native antibody signal sequence, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, lpp,or heat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,a factor leader (including Saccharomyces and Kluyveromyces α-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in WO 90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available.

(b) Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ, plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter.

(c) Selection of Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take upantibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS),thymidine kinase, metallothionein-I and —II, preferably primatemetallothionein genes, adenosine deaminase, ornithine decarboxylase,etc.

For example, cells transformed with the DHFR gene are identified byculturing the transformants in a culture medium containing methotrexate(Mtx), a competitive antagonist of DHFR. Under these conditions, theDHFR gene is amplified along with any other co-transformed nucleic acid.A Chinese hamster ovary (CHO) cell line deficient in endogenous DHFRactivity (e.g., ATCC CRL-9096) may be used.

Alternatively, cells transformed with the GS gene are identified byculturing the transformants in a culture medium containing L-methioninesulfoximine (Msx), an inhibitor of GS. Under these conditions, the GSgene is amplified along with any other co-transformed nucleic acid. TheGS selection/amplification system may be used in combination with theDHFR selection/amplification system described above.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody of interest, wild-type DHFR gene, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

(d) Promoter component

Expression and Cloning Vectors Generally Contain a Promoter that isRecognized by the host organism and is operably linked to nucleic acidencoding an antibody. Promoters suitable for use with prokaryotic hostsinclude the pho A promoter, β-lactamase and lactose promoter systems,alkaline phosphatase promoter, a tryptophan (trp) promoter system, andhybrid promoters such as the tac promoter. However, other knownbacterial promoters are suitable. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding an antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells can becontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40(SV40), or from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(e) Enhancer Element Component

Transcription of a DNA encoding an antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the antibody-encoding sequence, butis preferably located at a site 5′ from the promoter.

(f) Transcription Terminator Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

(g) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Envinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

Full length antibody, antibody fusion proteins, and antibody fragmentscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) that by itself showseffectiveness in tumor cell destruction. Full length antibodies havegreater half-life in circulation. Production in E. coli is faster andmore cost efficient. For expression of antibody fragments andpolypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et.al.), U.S. Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523(Simmons et al.), which describes translation initiation region (TIR)and signal sequences for optimizing expression and secretion. See alsoCharlton, Methods in Molecular Biology, Vol. 248, B. K. C. Lo, ed.,Humana Press, Totowa, N.J., pp. 245-254 (2003), describing expression ofantibody fragments in E. coli. After expression, the antibody may beisolated from the E. coli cell paste in a soluble fraction and can bepurified through, e.g., a protein A or G column depending on theisotype. Final purification can be carried out similar to the processfor purifying antibody expressed e.g., in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger. For a reviewdiscussing the use of yeasts and filamentous fungi for the production oftherapeutic proteins, see, e.g., Gerngross, Nat. Biotech. 22:1409-1414(2004).

Certain fungi and yeast strains may be selected in which glycosylationpathways have been “humanized,” resulting in the production of anantibody with a partially or fully human glycosylation pattern. See,e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describinghumanization of the glycosylation pathway in Pichia pastoris); andGerngross et al., supra.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frupperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to theinvention, particularly for transfection of Spodoptera frupperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco can also beutilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498,6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technologyfor producing antibodies in transgenic plants).

Vertebrate cells may be used as hosts, and propagation of vertebratecells in culture (tissue culture) has become a routine procedure.Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2). Other useful mammalian host cell lines include Chinese hamsterovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al., Proc. Natl.Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NSO andSp2/0. For a review of certain mammalian host cell lines suitable forantibody production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, 248:255-268 (2003).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(h) Culturing Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(xi) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, hydrophobic interactionchromatography, gel electrophoresis, dialysis, and affinitychromatography, with affinity chromatography being among one of thetypically preferred purification steps. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX′ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE′ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

In general, various methodologies for preparing antibodies for use inresearch, testing, and clinical are well-established in the art,consistent with the above-described methodologies and/or as deemedappropriate by one skilled in the art for a particular antibody ofinterest.

B. Selecting Biologically Active Antibodies

Antibodies produced as described above may be subjected to one or more“biological activity” assays to select an antibody with beneficialproperties from a therapeutic perspective. The antibody may be screenedfor its ability to bind the antigen against which it was raised. Forexample, for an anti-DR5 antibody (e.g., drozitumab), the antigenbinding properties of the antibody can be evaluated in an assay thatdetects the ability to bind to a death receptor 5 (DR5).

In another embodiment, the affinity of the antibody may be determined bysaturation binding; ELISA; and/or competition assays (e.g. RIA's), forexample.

Also, the antibody may be subjected to other biological activity assays,e.g., in order to evaluate its effectiveness as a therapeutic. Suchassays are known in the art and depend on the target antigen andintended use for the antibody.

To screen for antibodies which bind to a particular epitope on theantigen of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g. as described in Champe et al., J.Biol. Chem. 270:1388-1394 (1995), can be performed to determine whetherthe antibody binds an epitope of interest.

C. Preparation of the Formulations

Provided herein are formulations comprising a polysorbate and acyclodextrin that have reduced polysorbate degradation. In someembodiments the cyclodextrin is 2-hydroxypropyl-O-cyclodextrin(HP-β-CD). In some embodiments, the cyclodextrin is2-hydroxypropyl-α-cyclodextrin (HP-α-CD), 2-hydroxypropyl-γ-cyclodextrin(HP-γ-CD). In some embodiments, the cyclodextrin is β-cyclodextrin(β-CD). In some embodiments the cyclodextrin is sulfobutyl etherβ-cyclodextrin (SBE-β-CD). In some embodiments, the cyclodextrin isα-cyclodextrin (α-CD). In some embodiments, the cyclodextrin isγ-cyclodextrin (γ-CD). In some embodiments, the formulation comprises apolysorbate and a polyvinylpyrrolidone (PVP) and has reduced polysorbatedegradation. In some embodiments, the formulation further comprises apolypeptide. In some embodiments, the polysorbate is in the range ofabout 0.001% to about 15% or any range between these values. In certainembodiments the polysorbate is in the range of about 0.001% to about0.4%, 0.01% to about 0.4%, about 0.01% to about 0.3%, about 0.01% toabout 0.2%, about 0.01% to about 0.1%. In some embodiments, theformulation comprises about 0.001%, about 0.005% about 0.01%, about0.02% about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%,about 0.08%, about 0.09%, about 0.1%, about 0.4%, about 1%, about 5%, orabout 15% polysorbate. In some embodiments the polysorbate ispolysorbate 20. In some embodiments the polysorbate is polysorbate 40.In some embodiments the polysorbate is polysorbate 60. In someembodiments, the polysorbate is polysorbate 80.

In some embodiments polyvinalpyrrolidone is a class of polymericmolecules made from made from the monomer N-vinylpyrrolidone. In someembodiments the polyvinylpyrrolidone (PVP) is Povidone (soluble PVP). Insome embodiments the PVP is Povidone K12 (Approximate MW: 2.5 kDa). Insome embodiments the PVP is Povidone K15 (Approximate MW: 8 kDa). Insome embodiments the PVP is Povidone K17 (Approximate MW: 10 kDa). Insome embodiments the PVP is Povidone K25 (Approximate MW: 30 kDa). Insome embodiments the PVP is Povidone K30 (Approximate MW: 50 kDa). Insome embodiments the PVP is Povidone K60 (Approximate MW: 400 kDa). Insome embodiments the PVP is Povidone K90 (Approximate MW: 1,000 kDa). Insome embodiments the PVP is Povidone K120 (3,000 kDa). In someembodiments the PVP is Crospovidone (insoluble PVP). In some embodimentsthe PVP is Copovidone.

In some embodiments the cyclodextrin is in the range of about 0.5% toabout 30%. In some embodiments the cyclodextrin is in the range of about1% to about 25%, or about 5% to about 20%, or about 10% to about 15%. Infurther embodiments, the cyclodextrin is at a concentration of about0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, orabout 30%. In some embodiments the PVP is in the range of about 0.5% toabout 30%.

In some embodiments, the w/w ratio of cyclodextrin to polysorbate in theformulation is greater than about 37.5:1. In some embodiments, the w/wratio of cyclodextrin to polysorbate in the formulation is greater thanabout 50:1, greater than about 100:1, greater than about 150:1, greaterthan about 250:1, greater than about 750:1, greater than about 1000:1,or greater than about 3000:1. In some embodiments, the w/w ratio ofcyclodextrin to polysorbate is not between 67:1 and 1000:1. In someembodiments, the w/w ratio of PVP to polysorbate in the formulation isgreater than about 37.5:1.

In some embodiments, the aqueous formulation comprises a polypeptide ata concentration in the range from about 10 mg/mL to about 250 mg/mL orany range between these values. In some embodiments, the polypeptide isat a concentration greater than about 250 mg/mL. In some embodiments,the polypeptide is at a concentration in the range from any one of about10 mg/mL to 250 mg/mL, 50 mg/mL to 250 mg/mL, 100 mg/mL to 250 mg/mL,150 mg/mL to 250 mg/mL, 200 mg/mL to 250 mg/mL, 10 mg/mL to 200 mg/mL,50 mg/mL to 200 mg/mL, 100 mg/mL to 200 mg/mL, 150 mg/mL to 200 mg/mL,10 mg/mL to 150 mg/mL, 50 mg/mL to 150 mg/mL, 100 mg/mL to 150 mg/mL, 10mg/mL to 100 mg/mL, 50 mg/mL to 100 mg/mL, 10 mg/mL to 50 mg/mL or anyrange between these ranges.

In some embodiments, the aqueous formulation comprises an antibody. Insome embodiments, the antibody is directed to (VEGF); CD20; ox-LDL;ox-ApoB100; renin; a growth hormone, including human growth hormone andbovine growth hormone; growth hormone releasing factor; parathyroidhormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;insulin A-chain; insulin B-chain; proinsulin; follicle stimulatinghormone; calcitonin; luteinizing hormone; glucagon; clotting factorssuch as factor VIIIC, factor IX, tissue factor, and von Willebrandsfactor; anti-clotting factors such as Protein C; atrial natriureticfactor; lung surfactant; a plasminogen activator, such as urokinase orhuman urine or tissue-type plasminogen activator (t-PA); bombesin;thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and-beta; enkephalinase; RANTES (regulated on activation normally T-cellexpressed and secreted); human macrophage inflammatory protein(MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; receptors for hormonesor growth factors; protein A or D; rheumatoid factors; a neurotrophicfactor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3,-4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor suchas NGF-β; platelet-derived growth factor (PDGF); fibroblast growthfactor such as aFGF and bFGF; epidermal growth factor (EGF);transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des (1-3)-IGF-I (brain IGF-I),insulin-like growth factor binding proteins; CD proteins such as CD3,CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors;immunotoxins; a bone morphogenetic protein (BMP); an interferon such asinterferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs),e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;superoxide dismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrns such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of any of the above-listed polypeptides. Insome embodiments, the antibody is not an anti-CD20 antibody. In someembodiments, the formulation does not comprise an anti-CD20 antibody and0.2% polysorbate (e.g., polysorbate 80). In some embodiments, theformulation does not comprise 10% HP-γcyclodextrin and 0.03% polysorbate20. In some embodiments, the formulation does not comprise an anti-CD20antibody, 10% HP-γcyclodextrin and 0.03% polysorbate 20.

In some embodiments, the aqueous formulation further comprises one ormore excipients selected from the group consisting of a stabilizer, abuffer, a surfactant, and a tonicity agent. An aqueous formulation ofthe invention can be prepared in a pH-buffered solution. The buffer ofthis invention has a pH in the range from about pH 4.5 to about 9.0. Incertain embodiments the pH is in the range from about pH 4.5 to about7.0, in the range from about pH 4.5 to about 6.5, in the range fromabout pH 4.5 to about 6.0, in the range from about pH 4.5 to about 5.5,in the range from about pH 4.5 to about 5.0, in the range from about pH5.0 to about 7.0, in the range from about pH 5.5 to about 7.0, in therange from about pH 5.7 to about 6.8, in the range from about pH 5.8 toabout 6.5, in the range from about pH 5.9 to about 6.5, in the rangefrom about pH 6.0 to about 6.5, or in the range from about pH 6.2 toabout 6.5. In certain embodiments, the liquid formulation has a pH inthe range of about 4.7 to about 5.2, in the range of about 5.0 to 6.0,or in the range of about 5.2 to about 5.8. In certain embodiments of theinvention, the liquid formulation has a pH of 6.2 or about 6.2. Incertain embodiments of the invention, the liquid formulation has a pH of6.0 or about 6.0.

Examples of buffers that will control the pH within this range includeorganic and inorganic acids and salts thereof. For example, acetate(e.g., histidine acetate, arginine acetate, sodium acetate), succinate(e.g., histidine succinate, arginine succinate, sodium succinate),gluconate, phosphate, fumarate, oxalate, lactate, citrate, andcombinations thereof. The buffer concentration can be from about 1 mM toabout 600 mM, depending, for example, on the buffer and the desiredisotonicity of the formulation.

Additional surfactants can optionally be added to the aqueousformulation. Exemplary surfactants include nonionic surfactants such aspoloxamers (e.g. poloxamer 188, etc.). The amount of surfactant added issuch that it reduces aggregation of the formulated antibody and/orminimizes the formation of particulates in the formulation and/orreduces adsorption. For example, the surfactant may be present in theformulation in an amount from about 0.001% to about 0.5%, from about0.005% to about 0.2%, from about 0.01% to about 0.1%, or from about0.02% to about 0.06%, or about 0.03% to about 0.05%. In certainembodiments, the surfactant is present in the formulation in an amountof 0.04% or about 0.04%. In certain embodiments, the surfactant ispresent in the formulation in an amount of 0.02% or about 0.02%. In oneembodiment, the formulation does not comprise a surfactant.

Tonicity agents, sometimes known as “stabilizers” are present to adjustor maintain the tonicity of liquid in a composition. When used withlarge, charged biomolecules such as proteins and antibodies, they areoften termed “stabilizers” because they can interact with the chargedgroups of the amino acid side chains, thereby lessening the potentialfor inter- and intra-molecular interactions. Tonicity agents can bepresent in any amount between 0.1% to 25% by weight, or more preferablybetween 1% to 5% by weight, taking into account the relative amounts ofthe other ingredients. Preferred tonicity agents include polyhydricsugar alcohols, preferably trihydric or higher sugar alcohols, such asglycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

In some embodiments, the formulation is for in vivo administration. Insome embodiments, the formulation is sterile. The formulation may berendered sterile by filtration through sterile filtration membranes. Thetherapeutic formulations herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.The route of administration is in accordance with known and acceptedmethods, such as by single or multiple bolus or infusion over a longperiod of time in a suitable manner, e.g., injection or infusion bysubcutaneous, intravenous, intraperitoneal, intramuscular,intraarterial, intralesional or intraarticular routes, topicaladministration, inhalation or by sustained release or extended-releasemeans.

The aqueous formulations provided by the invention comprise apolypeptide, a polysorbate, and a cyclodextrin and show enhancedpolysorbate stability after a period of storage. In one embodiment, thepolysorbate stability is expressed as a relative percent of polysorbateremaining in a formulation after a period of storage. For example if aformulation that contains 0.1% polysorbate initially and contains 0.09%polysorbate after a period of storage, 10% of the polysorbate has beendegraded. In a further embodiment, the amount of polysorbate in asolution is determined by reverse phase ultra-performance liquidchromatography using evaporative light scattering detection (RP-ELSD)(Kim, J & Qiu, J. 2014, Analytica Chimica Acta 806:144-151). In someembodiments the concentration of polysorbate in a sample is determinedby comparing the sample results to a standard curve generated usingdifferent polysorbate concentrations.

In some embodiments, less than 5% of the polysorbate has degraded afterthe formulation is stored at about 1° C. to about 10° C. for about sixmonths, at least about 12 months, at least about 18 months, at leastabout 24 months, at least about 30 months, at least about 36 months, atleast about 42 months, or at least about 48 months. In some embodiments,less than 5% of the polysorbate has degraded after the formulation isstored at about 2° C. to about 8° C. for at least about six months, atleast about 12 months, at least about 18 months, at least about 24months, at least about 30 months, at least about 36 months, at leastabout 42 months, or at least about 48 months. In some embodiments, lessthan 5% of the polysorbate has degraded after the formulation is storedat about 4° C. to about 6° C. for at least about six months, at leastabout 12 months, at least about 18 months, at least about 24 months, atleast about 30 months, at least about 36 months, at least about 42months, or at least about 48 months.

In some embodiments, less than 1% of the polysorbate has degraded afterthe formulation is stored at about 1° C. to about 10° C. for at leastabout six months, at least about 12 months, at least about 18 months, atleast about 24 months, at least about 30 months, at least about 36months, at least about 42 months, or at least about 48 months. In someembodiments, less than 1% of the polysorbate has degraded after theformulation is stored at about 2° C. to about 8° C. for at least aboutsix months, at least about 12 months, at least about 18 months, at leastabout 24 months, at least about 30 months, at least about 36 months, atleast about 42 months, or at least about 48 months. In some embodiments,less than 1% of the polysorbate has degraded after the formulation isstored at about 4° C. to about 6° C. for at least about six months, atleast about 12 months, at least about 18 months, at least about 24months, at least about 30 months, at least about 36 months, at leastabout 42 months, or at least about 48 months.

In some embodiments, less than 0.1% of the polysorbate has degradedafter the formulation is stored at about 1° C. to about 10° C. for atleast about six months, at least about 12 months, at least about 18months, at least about 24 months, at least about 30 months, at leastabout 36 months, at least about 42 months, or at least about 48 months.In some embodiments, less than 0.1% of the polysorbate has degradedafter the formulation is stored at about 2° C. to about 8° C. for atleast about six months, at least about 12 months, at least about 18months, at least about 24 months, at least about 30 months, at leastabout 36 months, at least about 42 months, or at least about 48 months.In some embodiments, less than 0.1% of the polysorbate has degradedafter the formulation is stored at about 4° C. to about 6° C. for atleast about six months, at least about 12 months, at least about 18months, at least about 24 months, at least about 30 months, at leastabout 36 months, at least about 42 months, or at least about 48 months.

In some embodiments, less than 5% of the polysorbate has degraded afterthe formulation is stored at about 22° C. to about 28° C. for at leastabout one month, at least about two months, at least about three months,at least about four months, at least about five months, at least aboutsix months, at least about seven months, at least about eight months, atleast about nine months, at least about ten months, at least abouteleven months, or at least about twelve months. In some embodiments,less than 1% of the polysorbate has degraded after the formulation isstored at about 22° C. to about 28° C. for at least about one month, atleast about two months, at least about three months, at least about fourmonths, at least about five months, at least about six months, at leastabout seven months, at least about eight months, at least about ninemonths, at least about ten months, at least about eleven months, or atleast about twelve months. In some embodiments, less than 0.1% of thepolysorbate has degraded after the formulation is stored at about 22° C.to about 28° C. for at least about one month, at least about two months,at least about three months, at least about four months, at least aboutfive months, at least about six months, at least about seven months, atleast about eight months, at least about nine months, at least about tenmonths, at least about eleven months, or at least about twelve months.

In some embodiments, less than 5% of the polysorbate has degraded afterthe formulation is stored at about −15° C. to about −25° C. for at leastabout 12 months, at least about 18 months, at least about 24 months, atleast about 30 months, at least about 36 months, at least about 42months, at least about 48 months, at least about 54 months, at leastabout 60 months, at least about 66 months, or at least about 72 months.In some embodiments, less than 1% of the polysorbate has degraded afterthe formulation is stored at about −15° C. to about −25° C. for at leastabout 12 months, at least about 18 months, at least about 24 months, atleast about 30 months, at least about 36 months, at least about 42months, at least about 48 months, at least about 54 months, at leastabout 60 months, at least about 66 months, or at least about 72 months.In some embodiments, less than 0.1% of the polysorbate has degradedafter the formulation is stored at about −15° C. to about −25° C. for atleast about 12 months, at least about 18 months, at least about 24months, at least about 30 months, at least about 36 months, at leastabout 42 months, or at least about 48 months, at least about 54 months,at least about 60 months, at least about 66 months, or at least about 72months.

In some embodiments, the formulation is stored at about −8° C. to about−80° C. In some embodiments, the formulation is stored at about −20° C.,−40° C., −70° C., or −80° C.

The formulations provided by the invention herein are effective forreducing polysorbate degradation products such as visible andsub-visible particles. In one embodiment, visible particles are observedby placing the sample in a glass vial and rotating the sample in thepresence of a Tyndall light. In one embodiment, subvisible particles areanalyzed using a high accuracy (HIAC) particle counter. In someembodiments, a HIAC 9703 particle counter equipped with an HRDL-150detector and a 1 mL syringe can be used. In some embodiments,performance of the instrument can be verified with NIST-traceable 2 μmPolystyrene bead standards at 3000 counts/mL before each measurementsession. In some embodiments, the HIAC instrument may be configured to a10 mL/min flow rate, 0.1 mL tare volume, and 0.4 mL sample volume. Inparticular embodiments, the samples may be analyzed using 4 runs of 0.4mL sips, with the first run of each sample was discarded to preventmeasurement error due to sample carryover. Filter sizes of 2, 5, 10, 15,and 25 μm can be used for analysis.

In some embodiments, the formulation has less than about 10,000, about5,000, about 1,000, about 500, about 250, about 150, about 100, about50, or about 25 particles greater than 1.4μ in diameter per mL. In someembodiments, the formulation has less than about 10,000, about 5,000,about 1,000, about 500, about 250, about 150, about 100, about 50, orabout 25 particles greater than 2μ in diameter per mL. In someembodiments, the formulation has less than about 1250, about 150, about100, about 50, about 25, about 20, about 15, about 10, about 5, about 4,about 3, about 2, or about 1 particles greater than 5μ in diameter permL. In some embodiments, the formulation has less than about 250, about150, about 100, about 50, about 25, about 20, about 15, about 10, about5, about 4, about 3, about 2, or about 1 particles 10μ in diameter permL. In some embodiments, the formulation has less than about 250, about150, about 100, about 50, about 25, about 20, about 15, about 10, about5, about 4, about 3, about 2, or about 1 particles greater than 15μ indiameter per mL. In some embodiments, the formulation has less thanabout 250, about 150, about 100, about 50, about 25, about 20, about 15,about 10, about 5, about 4, about 3, about 2, or about 1 particlesgreater than 25μ in diameter per mL.

III. Administration of Formulations

The aqueous formulation is administered to a mammal in need of treatmentwith the protein (e.g., an antibody), preferably a human, in accord withknown methods, such as intravenous administration as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intravitreal,intra-articular, intrasynovial, intrathecal, ocular, oral, topical, orinhalation routes. In one embodiment, the aqueous formulation isadministered to the mammal by intravenous administration. For suchpurposes, the formulation may be injected using a syringe or via an IVline, for example. In one embodiment, the liquid formulation isadministered to the mammal by subcutaneous administration.

The appropriate dosage (“therapeutically effective amount”) of theprotein will depend, for example, on the condition to be treated, theseverity and course of the condition, whether the protein isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the protein, the type ofprotein used, and the discretion of the attending physician. The proteinis suitably administered to the patient at one time or over a series oftreatments and may be administered to the patient at any time fromdiagnosis onwards. The protein may be administered as the sole treatmentor in conjunction with other drugs or therapies useful in treating thecondition in question. As used herein the term “treatment” refers toboth therapeutic treatment and prophylactic or preventative measures.Those in need of treatment include those already with the disorder aswell as those in which the disorder is to be prevented. As used herein a“disorder” is any condition that would benefit from treatment including,but not limited to, chronic and acute disorders or diseases includingthose pathological conditions which predispose the mammal to thedisorder in question.

In a pharmacological sense, in the context of the invention, a“therapeutically effective amount” of a protein (e.g., an antibody)refers to an amount effective in the prevention or treatment of adisorder for the treatment of which the antibody is effective. As ageneral proposition, the therapeutically effective amount of the proteinadministered will be in the range of about 0.1 to about 50 mg/kg ofpatient body weight whether by one or more administrations, with thetypical range of protein used being about 0.3 to about 20 mg/kg,preferably about 0.3 to about 15 mg/kg, administered daily, for example.However, other dosage regimens may be useful. For example, a protein canbe administered at a dose of about 100 or 400 mg every 1, 2, 3, or 4weeks or is administered a dose of about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 15.0,or 20.0 mg/kg every 1, 2, 3, or 4 weeks. The dose may be administered asa single dose or as multiple doses (e.g., 2 or 3 doses), such asinfusions. The progress of this therapy is easily monitored byconventional techniques.

IV. Methods of Reducing Polysorbate Degradation

Provided herein are methods of reducing polysorbate degradation in anaqueous formulation containing polysorbate, comprising adding acyclodextrin to the formulation. Also provided herein are methods ofreducing the amount of visible and sub-visible particles in an aqueoussolution containing polysorbate, comprising adding a cyclodextrin to theformulation. The invention also includes a method to disaggregate andsolubilize polysorbate degradation products in an aqueous solutioncomprising adding a cyclodextrin to the formulation. In someembodiments, the formulation further comprises a polypeptide, a nucleicacid, a lipid and/or a carbohydrate.

Provided herein are methods of reducing polysorbate in an aqueousformulation comprising polyvinylpryrrolidone (PVP) and polysorbate. Alsoprovided herein are methods of reducing the amount of visible andsub-visible particles in an aqueous solution containing polysorbate,comprising adding PVP to the formulation. The invention also includes amethod to disaggregate and solubilize polysorbate degradation productsin an aqueous solution comprising adding PVP to the formulation. In someembodiments, the formulation further comprises a polypeptide, a nucleicacid, a lipid and/or a carbohydrate.

In some embodiments, the polysorbate is in the range of about 0.001% toabout 0.4% or any range between these values. In certain embodiments thepolysorbate is in the range of about 0.001% to about 0.4%, about 0.01%to about 0.4%, about 0.01% to about 0.3%, about 0.01% to about 0.2%, orabout 0.01% to about 0.1%. In some embodiments, the formulationcomprises about 0.001%, about 0.005%, about 0.01%, about 0.02% about0.03% about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%,about 0.09%, about 0.1%, or about, 0.4% polysorbate. In some embodimentsthe polysorbate is polysorbate 20. In some embodiments the polysorbateis polysorbate 40. In some embodiments the polysorbate is polysorbate60. In some embodiments, the polysorbate is polysorbate 80.

In some embodiments the cyclodextrin is added to a concentration ofabout 0.5% to about 30%. In some embodiments the cyclodextrin is in therange of about 1% to about 25%, about 5% to about 20%, or about 10% toabout 15%. In further embodiments, the cyclodextrin is added to aconcentration of about 0.5%, about 1%, about 5%, about 10%, about 15%,about 20%, about 25%, or about 30%. In some embodiments PVP is added toa concentration of about 0.5% to about 30%.

In some embodiments, the formulation does not comprise 10%HP-γcyclodextrin and 0.03% polysorbate 20. In some embodiments, theformulation does not comprise an anti-CD20 antibody, 10%HP-γcyclodextrin and 0.03% polysorbate 20.

In some embodiments, the resulting w/w ratio of cyclodextrin topolysorbate in the formulation is greater than about 37.5:1. In someembodiments, the resulting w/w ratio of cyclodextrin to polysorbate inthe formulation is greater than about 50:1, greater than about 100:1,greater than about 150:1, greater than about 250:1, greater than about750 to 1, greater than about 1000:1, or greater than about 3000:1. Insome embodiments, the resulting w/w ratio of cyclodextrin to polysorbatein the formulation is not in between 67:1 and 1000:1. In someembodiments, the resulting w/w ratio of PVP to polysorbate in theformulation is greater than about 37.5:1. In some embodiments, theresulting ratio of PVP to polysorbate in the formulation is 250:1.

In some embodiments the cyclodextrin is 2-hydroxypropyl-β-cyclodextrin(HP-β-CD). In some embodiments, the cyclodextrin is2-hydroxypropyl-α-cyclodextrin (HP-α-CD), 2-hydroxypropyl-γ-cyclodextrin(HP-γ-CD). In some embodiments, the cyclodextrin is sulfobutyl etherβ-cyclodextrin (SBE-β-CD) In some embodiments, the cyclodextrin isβ-cyclodextrin (β-CD). In some embodiments, the cyclodextrin isα-cyclodextrin (α-CD). In some embodiments, the cyclodextrin isγ-cyclodextrin (γ-CD).

In some embodiments, the aqueous formulation comprises a polypeptide ata concentration in the range from 10 mg/mL to 250 mg/mL. In someembodiments, the polypeptide is at a concentration at above 250 mg/mL.In some embodiments, the polypeptide is at a concentration in the rangefrom 30 mg/mL to 150 mg/mL, 50 mg/mL to 150 mg/mL, or 100 to 150 mg/mL.

In some embodiments, the aqueous formulation comprises an antibody. Insome embodiments, the antibody is directed to (VEGF); CD20; ox-LDL;ox-ApoB100; renin; a growth hormone, including human growth hormone andbovine growth hormone; growth hormone releasing factor; parathyroidhormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;insulin A-chain; insulin B-chain; proinsulin; follicle stimulatinghormone; calcitonin; luteinizing hormone; glucagon; clotting factorssuch as factor VIIIC, factor IX, tissue factor, and von Willebrandsfactor; anti-clotting factors such as Protein C; atrial natriureticfactor; lung surfactant; a plasminogen activator, such as urokinase orhuman urine or tissue-type plasminogen activator (t-PA); bombesin;thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and-beta; enkephalinase; RANTES (regulated on activation normally T-cellexpressed and secreted); human macrophage inflammatory protein(MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; receptors for hormonesor growth factors; protein A or D; rheumatoid factors; a neurotrophicfactor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3,-4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor suchas NGF-β; platelet-derived growth factor (PDGF); fibroblast growthfactor such as aFGF and bFGF; epidermal growth factor (EGF);transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des (1-3)-IGF-I (brain IGF-I),insulin-like growth factor binding proteins; CD proteins such as CD3,CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors;immunotoxins; a bone morphogenetic protein (BMP); an interferon such asinterferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs),e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;superoxide dismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrns such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of any of the above-listed polypeptides. Insome embodiments, the antibody is not an anti-CD20 antibody.

In some embodiments, one or more excipients selected from the groupconsisting of a stabilizer, a buffer, a surfactant, and a tonicity agentare included in the aqueous formulation. The method the invention can becarried out prepared in a pH-buffered solution. The buffer of thisinvention has a pH in the range from about pH 4.5 to about 9.0. Incertain embodiments the pH is in the range from about pH 4.5 to about7.0, in the range from about pH 4.5 to about 6.6, in the range fromabout pH 4.5 to about 6.0, in the range from about pH 4.5 to about 5.5,in the range from about pH 4.5 to about 5.0, in the range from about pH5.0 to about 7.0, in the range from about pH 5.5 to about 7.0, in therange from about pH 5.7 to about 6.8, in the range from about pH 5.8 toabout 6.5, in the range from about pH 5.9 to about 6.5, in the rangefrom about pH 6.0 to about 6.5, or in the range from about pH 6.2 toabout 6.5. In certain embodiments, the liquid formulation has a pH inthe range of about 4.7 to about 5.2, in the range of about 5.0 to 6.0,or in the range of about 5.2 to about 5.8. In certain embodiments of theinvention, the liquid formulation has a pH of 6.2 or about 6.2. Incertain embodiments of the invention, the liquid formulation has a pH of6.0 or about 6.0.

Examples of buffers that will control the pH within this range includeorganic and inorganic acids and salts thereof. For example, acetate(e.g., histidine acetate, arginine acetate, sodium acetate), succinate(e.g., histidine succinate, arginine succinate, sodium succinate),gluconate, phosphate, fumarate, oxalate, lactate, citrate, andcombinations thereof. The buffer concentration can be from about 1 mM toabout 600 mM, depending, for example, on the buffer and the desiredisotonicity of the formulation.

Additional surfactants can optionally be added to the aqueousformulation. Exemplary surfactants include nonionic surfactants such aspoloxamers (e.g. poloxamer 188, etc.). The amount of surfactant added issuch that it reduces aggregation of the formulated antibody and/orminimizes the formation of particulates in the formulation and/orreduces adsorption. For example, the surfactant may be present in theformulation in an amount from about 0.001% to about 0.5%, from about0.005% to about 0.2%, from about 0.01% to about 0.1%, from about 0.02%to about 0.06%, or from about 0.03% to about 0.05%. In certainembodiments, the surfactant is present in the formulation in an amountof 0.04% or about 0.04%. In certain embodiments, the surfactant ispresent in the formulation in an amount of 0.02% or about 0.02%. In oneembodiment, the formulation does not comprise a surfactant.

The method may involve the use of tonicity agents, sometimes known as“stabilizers” to adjust or maintain the tonicity the aqueousformulation. When used with large, charged biomolecules such as proteinsand antibodies, they are often termed “stabilizers” because they caninteract with the charged groups of the amino acid side chains, therebylessening the potential for inter- and intra-molecular interactions.Tonicity agents can be present in any amount between 0.1% to 25% byweight, or more preferably between 1% to 5% by weight, taking intoaccount the relative amounts of the other ingredients. Preferredtonicity agents include polyhydric sugar alcohols, preferably trihydricor higher sugar alcohols, such as glycerin, erythritol, arabitol,xylitol, sorbitol and mannitol.

In some embodiments, the method results in less than 5% of thepolysorbate being degraded after the formulation is stored at about 1°C. to about 10° C. for at least about six months, at least about 12months, at least about 18 months, at least about 24 months, at leastabout 30 months, at least about 36 months, at least about 42 months, orat least about 48 months. In some embodiments, less than 5% of thepolysorbate has degraded after the formulation is stored at about 2° C.to about 8° C. for at least about six months at least about 12 months,at least about 18 months, at least about 24 months, at least about 30months, at least about 36 months, at least about 42 months, or at leastabout 48 months. In some embodiments, the method results in less than 5%of the polysorbate being degraded after the formulation is stored atabout 4° C. to about 6° C. for at least about six months, at least about12 months, at least about 18 months, at least about 24 months, at leastabout 30 months, at least about 36 months, at least about 42 months, orat least about 48 months.

In some embodiments, the method results in less than 1% of thepolysorbate being degraded after the formulation is stored at about 1°C. to about 10° C. for at least about six months, at least about 12months, at least about 18 months, at least about 24 months, at leastabout 30 months, at least about 36 months, at least about 42 months, orat least about 48 months. In some embodiments, the method results inless than 1% of the polysorbate being degraded after the formulation isstored at about 2° C. to about 8° C. for at least about six months, atleast about 12 months, at least about 18 months, at least about 24months, at least about 30 months, at least about 36 months, at leastabout 42 months, or at least about 48 months. In some embodiments, themethod results in less than 1% of the polysorbate has degraded after theformulation is stored at about 4° C. to about 6° C. for at least aboutsix months, at least about 12 months, at least about 18 months, at leastabout 24 months, at least about 30 months, at least about 36 months, atleast about 42 months, or at least about 48 months.

In some embodiments, the method results in less than 0.1% of thepolysorbate being degraded after the formulation is stored at about 1°C. to about 10° C. for at least about six months, at least about 12months, at least about 18 months, at least about 24 months, at leastabout 30 months, at least about 36 months, at least about 42 months, orat least about 48 months. In some embodiments, the method results inthan 0.1% of the polysorbate being degraded after the formulation isstored at about 2° C. to about 8° C. for at least about six months, atleast about 12 months, at least about 18 months, at least about 24months, at least about 30 months, at least about 36 months, at leastabout 42 months, or at least about 48 months. In some embodiments, themethod results in less than 0.1% of the polysorbate being degraded afterthe formulation is stored at about 4° C. to about 6° C. for at leastabout six months, at least about 12 months, at least about 18 months, atleast about 24 months, at least about 30 months, at least about 36months, at least about 42 months, or at least about 48 months.

In some embodiments, the method results in less than 5% of thepolysorbate being degraded after the formulation is stored at about 22°C. to about 28° C. for at least about one month, at least about twomonths, at least about three months, at least about four months, atleast about five months, at least about six months, at least about sevenmonths, at least about eight months, at least about nine months, atleast about ten months, at least about eleven months, or at least abouttwelve months. In some embodiments, the method results in less than 1%of the polysorbate being degraded after the formulation is stored atabout 22° C. to about 28° C. for at least about one month, at leastabout two months, at least about three months, at least about fourmonths, at least about five months, at least about six months, at leastabout seven months, at least about eight months, at least about ninemonths, at least about ten months, at least about eleven months, or atleast about twelve months. In some embodiments, the method results inless than 0.1% of the polysorbate being degraded after the formulationis stored at about 22° C. to about 28° C. for at least about one month,at least about two months, at least about three months, at least aboutfour months, at least about five months, at least about six months, atleast about seven months, at least about eight months, at least aboutnine months, at least about ten months, at least about eleven months, orat least about twelve months.

In some embodiments, less than 5% of the polysorbate has degraded afterthe formulation is stored at about −15° C. to about −25° C. for at leastabout 12 months, at least about 18 months, at least about 24 months, atleast about 30 months, at least about 36 months, at least about 42months, at least about 48 months, at least about 54 months, at leastabout 60 months, at least about 66 months, or at least about 72 months.In some embodiments, the method results in less than 1% of thepolysorbate being degraded after the formulation is stored at about −15°C. to about −25° C. for at least about 12 months, at least about 18months, at least about 24 months, at least about 30 months, at leastabout 36 months, at least about 42 months, at least about 48 months, atleast about 54 months, at least about 60 months, at least about 66months, or at least about 72 months. In some embodiments, the methodresults in less than 0.1% of the polysorbate being degraded after theformulation is stored at about −15° C. to about −25° C. for at leastabout 12 months, at least about 18 months, at least about 24 months, atleast about 30 months, at least about 36 months, at least about 42months, or at least about 48 months, at least about 54 months, at leastabout 60 months, at least about 66 months, or at least about 72 months.

The methods provided by the invention are effective in reducing thenumber of sub-visible and visible particles. In some embodiments, lessthan about 10,000, about 5,000, about 1,000, about 500, about 250, about150, about 100, about 50, or about 25 particles greater than 1.4μ indiameter are formed per mL. In some embodiments, the formulation hasless than about 10,000, about 5,000, about 1,000, about 500, about 250,about 150, about 100, about 50, or about 25 particles greater than 2μ indiameter per mL. In some embodiments, less than about 1250, about 150,about 100, about 50, about 25, about 20, about 15, about 10, about 5,about 4, about 3, about 2, or about 1 particles greater than 5μ indiameter are formed per mL. In some embodiments, less than about 250,about 150, about 100, about 50, about 25, about 20, about 15, about 10,about 5, about 4, about 3, about 2, or about 1 particles 10μ in diameterare formed per mL. In some embodiments, less than about 250, about 150,about 100, about 50, about 25, about 20, about 15, about 10, about 5,about 4, about 3, about 2, or about 1 particles greater than 15μ indiameter are formed per mL. In some embodiments, less than about 250,about 150, about 100, about 50, about 25, about 20, about 15, about 10,about 5, about 4, about 3, about 2, or about 1 particles greater than25μ in diameter are formed per mL.

A method of resolubilizing polysorbate degradation products in aformulation is also provided herein. In some embodiments, the number ofparticles greater than 1.4μ present in the formulation is reduced by100, 1000, 2000, 5000, or 10000 fold after adding polysorbate. In someembodiments, the number of particles greater than 2μ in diameter presentin the formulation is reduced by 100, 1000, 2000, 5000, or 10000 foldafter adding polysorbate. In some embodiments, the number of particlesgreater than 5μ in diameter present in the formulation is reduced by100, 1000, 2000, 5000, or 10000 fold after adding polysorbate. In someembodiments, the number of particles greater than 10μ in diameterpresent in the formulation is reduced by 100, 1000, 2000, 5000, or 10000fold after adding polysorbate. In some embodiments, the number ofparticles greater than 15μ in diameter present in the formulation isreduced by 100, 1000, 2000, 5000, or 10000 fold after addingpolysorbate. In some embodiments, the number of particles greater than25μ in diameter present in the formulation is reduced by 100, 1000,2000, 5000, or 10000 fold after adding polysorbate.

V. Articles of Manufacture

In another embodiment of the invention, an article of manufacture isprovided comprising a container which holds the liquid formulation ofthe invention and optionally provides instructions for its use. Suitablecontainers include, for example, bottles, vials and syringes. Thecontainer may be formed from a variety of materials such as glass orplastic. An exemplary container is a 3-20 cc single use glass vial.Alternatively, for a multidose formulation, the container may be 3-100cc glass vial. The container holds the formulation and the label on, orassociated with, the container may indicate directions for use. Thearticle of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

VI. Kits

In another embodiment of the invention, kits for reducing polysorbatedegradation are provided. In some embodiments, the invention provideskit for use in reducing polysorbate degradation by the methods describedherein. In some embodiments, a kit comprising any of the formulationsprovided herein is provided. In one embodiment, such kits comprise acontainer of an aqueous formulation of therapeutic peptide or antibodyand a solution of a cyclodextrin that can be added to the aqueousformulation, wherein the ratio of cyclodextrin to polysorbate is greaterthan 37.5:1. In one embodiment, such kits comprise a container of anaqueous formulation of therapeutic peptide or antibody and a solution ofpolyvinylpyrrolidone (PVP) that can be added to the aqueous formulation,wherein the ratio of PVP to polysorbate is greater than 37.5:1.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Materials and Methods

Materials

Hydroxypropyl-β-cyclodextrin (HP-β-CD) as was obtained from Ashland Inc.(Ashland, Ky.) as Cavitron W7 HP5 Pharma. Sulfobutyletherβ-cyclodextrin(SBE-β-CD) was obtained from Ligand Pharmaceuticals (La Jolla, Calif.)as Captisol. Hydroxypropyl-alpha-cyclodextrin (HP-α-CD),hydroxypropyl-γ-cyclodextrin (HP-γ-CD), polyethylene glycol (PEG 1500),and methionine were obtained from Sigma Aldrich (St. Louis, Mo.).Polyvinylpyrrolidone (PVP) was obtained as KollidonPovidone K-157 fromSpectrum Chemical (Gardena, Calif.). Polysorbate 20 (PS20) was obtainedfrom Croda Inc. (New Castle, Del.). Porcine pancreatic lipase (PPL),lipoprotein lipase from Burkholderia sp. (LPL), Candida antarcticalipase B (CALB), rabbit liver esterase (RLE), and2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) were obtainedfrom Sigma Aldrich Inc. (St. Louis, Mo.).

Determination of Subvisible Particle Counts by HIAC

Subvisible particles were measured using a HIAC 9703 particle counterequipped with an HRDL-150 detector and a 1 mL syringe. Performance ofthe instrument was verified with NIST-traceable 2 μm Polystyrene beadstandards at 3000 counts/mL before each measurement session. The HIACinstrument was configured to a 10 mL/min flow rate, 0.1 mL tare volume,and 0.4 mL sample volume. The samples were analyzed using 4 runs of 0.4mL sips, with the first run of each sample was discarded to preventmeasurement error due to sample carryover. Results were reported as theaverage values for 1.4, 2, 5, 10, 15, and 25, 50 μm analysis filtersizes.

Determination of Polysorbate Concentration

Polysorbate concentration was determined using reverse phaseultra-performance liquid chromatography using evaporative lightscattering detection (RP-ELSD). Samples were analyzed using an Agilent1100 series high performance liquid chromatography system (HPLC) fittedwith a Waters Oasis MAX cartridge column (20×2.1 mm, 30 μm particlesize). The HPLC system was set up with a switching valve directingcolumn flow-through to either waste or a Varian 380-LC evaporative lightscattering detector set to 100° C. The mobile phases consisted of 2%formic acid in water (Pump A) and 2% formic acid in isopropanol (PumpB). The pump gradient was isocratic at 10% pump B during equilibration,linear to 20% pump B for 1 minute, isocratic at 20% pump B for 2.4minutes, linear to 100% pump B for 0.1 minutes, isocratic at 100% pump Bfor 1.1 minutes, linear to 10% pump B for 0.1 minutes, and finallyisocratic at 10% pump B for 1.9 minutes. The switching valve directedcolumn flow-through to waste at the beginning of every injection, andthen directed flow to the detector after 2.4 minutes until the end ofthe gradient. In order to quantitate the PS20 concentration, a standardcurve was generated by injecting 20 μL of solutions containing between0% w/v and 0.4% w/v PS20. For excipients which affected the migrationtime of PS20 through the column, PS20 standard curve solutionscontaining the relevant excipient were included in the analysis tofacilitate accurate quantitation.

Visible Particle Imaging by Seidenader

Vials were inspected for visible particulates using a Seidenader V90-Tvisual inspection unit (Markt Schwaben, Germany) with vial carriagetilted to 60 degrees. Visible particle inspection of samples wasperformed by placing the glass vials in the holder and rotating in thepresence of a Tyndall light directed through the bottom of the vial.Pre-rotation (i.e., fast rotation) was first performed to agitate theliquids, suspend, and circulate particles. Following rotation andillumination, the particles are in motion and the light causesreflections of the particles that make them visible (i.e., Tyndalleffect). Visible particles were then observed using a magnifying lens.Videos and photographs of visible particles were obtained using aSamsung (seoul, South Korea) Galaxy device.

Determination of Turbidity

Turbidity was determined using UV spectroscopy. The UV absorbance ofeach sample was measured by recording the absorbance at 279 nm and 320nm in a quartz cuvette with 1-cm path length on an Agilent 8453spectrophotometer using Chemstation software (Agilent Technologies,Santa Clara, Calif.).

Determination of Protein Concentration

Protein concentration was measured by The UV absorbance of each samplewas measured by recording the average absorbance between 340 nm and 360nm in a quartz cuvette with 1-cm path length on an Agilent 8453spectrophotometer using Chemstation software (Agilent Technologies,Santa Clara, Calif.). The UV concentration determination was calculatedby using the experimentally determined absorptivities for each protein.The measurements were blanked against the appropriate buffers.

Example 1: Oxidative Degradation of Polysorbate

The ability of cyclodextrins to inhibit oxidative PS20 degradation wasevaluated using 2,2′-Azobisisobutyramidinium (AAPH), which has beenshown previously to degrade PS20 ( Borisov et al., J. Pharm. Sci.104:1005-1018 (2015)). To evaluate the ability of cyclodextrins toinhibit oxidation of PS20, samples containing either 15% (w/v) HP-β-CDor 15% (w/v) sucrose were compared to control samples (without anyadditional excipients). Polysorbate 20 degradation was determined byRP-ELSD for samples oxidized with 5 mM AAPH at 40° C. for 24 hourscontaining no excipient (control), 15% (w/v) sucrose, and 15% (w/v)HP-β-CD.

As shown in FIG. 1, the data demonstrate that both HP-β-CD and sucrosedecrease the amount of PS20 degradation. Following incubation with AAPH,a decrease of 17.9 in relative percent PS20 degradation was observed inthe control sample. Conversely, smaller decreases of 9.8 and 3.6 inpercent PS20 degradation were observed for samples containing 15% (w/v)sucrose and HP-β-CD, respectively.

Example 2: Inhibitory Effects of HP-β-CD on the Enzymatic Degradation ofPolysorbate 20

The effect of 15% HP-β-CD on the enzymatic degradation of PS20 wasmeasured. Samples of 0.02% PS20 in pH 5.5 buffer containing either noadditional excipient, 15% Sucrose, or 15% HP-β-CD were digested witheach of the enzymes porcine pancreatic lipase (PPL), lipoprotein lipase(LPL), Candida Antarctica lipase (CALB,) and rabbit liver esterase (RLE)at room temperature. PPL samples were digested with 15 μg/mL enzyme for4.5 hours. LPL samples were digested with 70 μg/mL enzyme for 5 hours.CALB samples were digested with 0.1 mg/mL immobilized enzyme for 1 hour.RLE samples were digested with 15 μg/mL enzyme for 5 hours. Alldigestions were conducted at room temperature.

PPL digestion was stopped by heat inactivation in an 85° C. water bathfor 30 minutes. LPL and RLE digestion could not be stopped by heatinactivation, so the samples were analyzed immediately for PS20 content.CALB digestion was stopped by filtering out the immobilized enzymebeads. In order to permit particle formation, CALB samples were placedat 5° C. overnight. RLE and LPL samples were frozen at −20° C. overnightto impede enzymatic activity and were then thawed over ice immediatelyprior to particle analysis.

As described above, all samples were analyzed for PS20 content by highperformance liquid chromatography (Agilent 1100 series) with an inlineevaporative light scattering detector (Varian 380-LC series). Visibleparticle inspection was conducted on a Seidenader visual inspectioninstrument. Subvisible particle analysis was conducted on a HIAC 9703particle counter.

Samples treated with CALB, RLE, and LPL showed 59.2%-68.3% reductions inPS20 (FIG. 2). Conversely, samples that contain 15% of HP-β-CD showedsignificant inhibition of PS20 degradation. Specifically, for thesesamples, PS20 concentration was reduced by 14.3%-37.4% (FIG. 2 and Table1).

TABLE 1 Enzymatic Degradation of Polysorbate 20 by CALB, LPL, and RLEHP-β-CD Polysorbate 20 Enzyme (% w/v) (Relative %) Candida Antarctica 035.3% Lipase B (CALB) 15 85.7% Lipoprotein Lipase 0 40.7% (LPL) 15 77.5%Rabbit Liver 0 31.7% Esterase (RLE) 15 62.7%

Additionally, HIAC data demonstrates that 15% (w/v) HP-β-CD reduces theformation of subvisible particulates (SVP). Following enzymaticdegradation with multiple enzymes (CALB, LPL, and RLE), fewer SVP/mLwere observed for all particle size classifications (≥2, ≥5, ≥10, and≥25 micron particles) when 15% (w/v) HP-β-CD was included in the sample.Similar results were obtained for LPL and RLE; however, the very smallquantities of ≥10 and ≥≥25 micron particles preclude interpretation of≥10 and ≥25 micron particle count measurements (FIGS. 3A-3D).

These findings demonstrate that HP-β-CD, a representative cyclodextrincomplex, is capable of inhibiting enzymatic degradation of PS20 bymultiple enzymes. Without being bound by theory, this may suggest thatthe primary mechanism of protection of PS20 by cyclodextrin moleculesinvolves a direct interaction between the cyclodextrin and thepolysorbate molecules.

Example 3: Inhibitory Effects of HP-β-CD on the Enzymatic Degradation ofPolysorbate 80

The effect of 15% (w/v) HP-β-CD on the enzymatic degradation of PS80 wasmeasured. Samples of 0.02% (w/v) PS80 in pH 5.5 buffer containing 0% and15% (w/v) HP-β-CD were digested with 15 μg/mL porcine pancreatic lipase(PPL) for 5 hours at room temperature. PPL digestion was stopped by heatinactivation in an 85° C. water bath for 30 minutes.

Following PPL digestion, the RP-ELSD demonstrates a decrease ofapproximately 19 relative percent in PS80 (FIG. 4). Conversely, adecrease of approximately 4% PS80 degradation was observed in samplesthat contain 15% (w/v) of HP-β-CD.

Additionally, HIAC data demonstrates that 15% (w/v) HP-β-CD reduces theaverage (n=3) quantity of subvisible particulates (SVP). Followingenzymatic degradation with PPL, fewer SVP/mL were observed for allparticle size classifications (≥1.4, ≥2, ≥5, ≥10, and ≥25 micronparticles) when 15% (w/v) HP-β-CD was included in the sample (FIGS.5A-5F).

These findings demonstrate that HP-β-CD, a representative cyclodextrincomplex, is capable of inhibiting enzymatic degradation of PS80. Theseresults suggest that the ability of cyclodextrins to reduce polysorbatedegradation, reduce particle formation, and solubilize existingparticles is generally applicable to the class of polysorbate molecules(e.g., PS20, PS40, PS60, PS80, etc.). Without being bound by theory,this may suggest that the primary mechanism of protection ofpolysorbates by cyclodextrin molecules involves a direct interactionbetween the cyclodextrin and conserved chemical structure subunits(e.g., fatty acids) that comprise all polysorbate molecules.

Example 4: Kinetics of Enzymatic Degradation of Polysorbate 20

To evaluate the kinetics of enzymatic PS20 degradation, samples weredigested with 15 μg/mL of PPL at room temperature in protein-freesamples containing 0.02% (w/v) PS20 and 15% sucrose, 15% HP-β-CD, or 15%HP-α-CD were digested using PPL incubated for 180 hours at about 25° C.As described above, all samples were analyzed for PS20 content by highperformance liquid chromatography (Agilent 1100 series) with an inlineevaporative light scattering detector (Varian 380-LC series). Subvisibleparticle analysis was conducted on a HIAC 9703 particle counter.

The 15% sucrose curve in FIG. 6 shows that PS20 degradation can bedescribed by a one phase exponential decay. The half-life is 32.91 hourswith a plateau of 44% (FIG. 6). These degradation kinetics support theuse of a 4.5 hour digestion model using PPL at 25° C. for 4.5 hours forsubsequent studies.

Example 5: Inhibitory Effects of Cyclodextrin and Other Excipients onthe Enzymatic Degradation of Polysorbate

Several excipients were tested for their ability to protect PS20 againstenzymatic hydrolysis including HP-α-CD, HP-β-CD, HP-γ-CD, SBE-β-CD, PVP,PEG 1500, sucrose, and methionine. Solutions of each excipient in pH 5.5buffer were prepared containing a final concentration of 0.02% PS20after enzyme addition. The concentration of HP-α-CD, HP-β-CD, HP-γ-CD,and SBE-β-CD in the excipient solutions was 106 mM. The methioninesample contained 10 mg/mL methionine due to solubility limitations. Theremaining excipients (PVP, PEG 1500, sucrose) were added to 15% w/v tomatch the % w/v of HP-β-CD. Samples were digested with 15 μg/mL PPLenzyme for 4.5 hours at room temperature, followed by 30 minutes of heatinactivation at 85° C. Each sample was analyzed for PS20 concentrationusing high performance liquid chromatography with an inline evaporativelight scattering detector. Samples were then placed at 5° C. overnightto allow for the formation of particles and were analyzed for visibleand subvisible particles as described previously.

The RP-ELSD results demonstrate that the excipient class is importantfor determining the extent of enzymatic PS20 degradation (FIG. 7).Following enzymatic digestion, the control sample (i.e., no excipient)had a 58 percent decrease in PS20 degradation. An equivalent decrease of58 percent PS20 was observed for samples containing 15% (w/v) sucrose.These findings demonstrate sucrose, an acyclic disaccharide (i.e.,sucrose) does not have an inhibitory effect on enzymatic PS20degradation. The inhibitory effects of cyclodextrin cannot solely beattributed to mass dilution effects because the results demonstrate thatequivalent masses of other excipients (e.g., sucrose) do not mitigatecatalytic polysorbate degradation.

Similarly, samples containing methionine did not have an inhibitoryeffect on enzymatic PS20 degradation or SVP formation (FIGS. 7 and8A-8D). Presumably, methionine would prevent oxidative PS20 degradationbut would not prevent enzymatic PS20 degradation. Without being bound bytheory, the mechanism of PS20 degradation reproduced in this experimentis likely hydrolytic and independent of oxidative PS20 degradation.

The results demonstrate that PEG has a small inhibitory effect on PS20degradation relative to the control sample. A decrease of 51% PS20 wasobserved for samples containing 5% (w/v) PEG 1500.

The cyclodextrin molecules evaluated (HP-α-CD, HP-β-CD, and SBE-β-CD)all had significant inhibitory effects on enzymatic PS20 degradation andSVP formation (FIGS. 7 and 8A-8D). Interestingly, the number of sugarsubunits may be important in determining the extent of inhibition by thecyclodextrin. For example, decreases of 1% and 7% PS20 were observed forHP-α-CD and HP-β-CD, respectively.

Further studies were performed to evaluate in the importance of HP-α-CD,HP-β-CD, and HP-γ-CD to further understand the importance ofcyclodextrin ring size. The data shown in FIG. 9 indicate that nosignificant PS20 degradation was observed for samples containing HP-α-CDand HP-β-CD; conversely, ˜82% in PS20 degradation was observed insamples containing 15% HP-γ-CD. Similarly, significant decreases in PS20concentration were observed in the control samples (i.e., no excipientand 15% sucrose) (FIG. 9). These findings demonstrate that the smallercyclodextrins HP-α-CD (Cavity Diameter: 4.7-5.2 Å; Cavity Volume: 174Å³) and HP-β-CD (Cavity Diameter: 6.0-6.5 Å; Cavity Volume: 262 Å³) aremore effective inhibitors of polysorbate 20 degradation than HP-γ-CD(Cavity Diameter: 7.5-8.3 Å; Cavity Volume: 472 Å³).

The dimensions and volume of the cavity for each cyclodextrin maydetermine their effectiveness as molecular inhibitors of enzymatic PS20degradation rather than the physicochemical properties of eachcyclodextrin. Without being bound by theory, this finding suggests thatthe mechanism of protection may involve a host-guest complexationbetween the cyclodextrins and the polysorbate 20 reactive site.

Example 6: Solubilization of Visible and Subvisible Particles Related toEnzymatic Polysorbate 20 Degradation by Cyclodextrins and OtherExcipients

Several excipients were tested for their ability to solubilize particlesproduced as a result of enzymatic PS20 degradation. Solutions ofconcentrated HP-α-CD, HP-β-CD, HP-γ-CD, SBE-β-CD, PVP, PEG 1500,sucrose, and methionine were prepared in triplicate in pH 5.5 buffer.Particles from enzymatic PS20 degradation were prepared. Three solutionsof 0.05% PS20 in pH 5.5 buffer were enzymatically degraded with 37.5μg/mL PPL for 4.5 hours at room temperature, followed by 30 minutes ofheat inactivation at 85° C. The degraded PS20 solutions were placed at5° C. to allow for crystallization of particles.

After particle formation in the degraded PS20 solutions, the remainingsample preparation was conducted in a 5° C. cold room to prevent thePS20 derived particles from dissolving. Each degraded PS20 solution wasdivided into 11 aliquots and concentrated excipient was spiked into eachthe aliquots. The final concentration of excipient in each sample was asfollows: 5% sucrose, 10 mg/mL methionine, 5% PVP, 5% PEG 1500, 15%HP-β-CD, 5% HP-β-CD, 0.5% HP-β-CD, 35.5 mM HP-α-CD, 35.5 mM HP-γ-CD, and35.5 mM SBE-β-CD. After the addition of each excipient, samples wereleft at 5° C. overnight. The following day, vials were inspected forvisible particles under a Seidenader visual inspection instrument.Subvisible particle counts were measured using a HIAC 9703 particlecounter.

The results demonstrate that cyclodextrins (HP-α-CD, HP-β-CD, andHP-γ-CD) were able to significantly reduce the amount of SVP relative tothe control and other excipient samples (FIGS. 10A and 10B). Theseresults establish that in addition to preventing enzymatic polysorbatedegradation, cyclodextrins can also solubilize the PS20 degradants thatresult from enzymatic digestion of polysorbate 20.

Additionally, photographs were taken immediately before and afteraddition of 15% (w/v) HP-β-CD. The photographs depict the solubilizationof PS20-related visible particles before (FIG. 11A) and after additionof 15% (w/v) HP-β-CD (FIG. 11B). The immediate solubilization of visibleparticles represented in the photographs provides compelling evidencethat cyclodextrins can increase the solubility of visible and subvisibleparticles associated with PS20 degradation.

Example 7: Solubilization of Visible and Subvisible Particles Related toOxidative Polysorbate 20 Degradation by Cyclodextrins

Different concentrations of HP-β-CD were tested for their ability tosolubilize particles produced as a result of oxidative PS20 degradation.Solutions of concentrated HP-β-CD were prepared in triplicate in pH 5.5buffer. Protein-free samples containing 0.02% (w/v) PS20 were stored for27 months at 5° C., resulting in oxidative PS20 degradation and theformation of visible and subvisible particles related to PS20degradation products. Each degraded PS20 solution was divided into 3aliquots and concentrated excipient was spiked into each the aliquots.The final concentration of excipient in each sample was as follows: 0%(w/v) HP-β-CD (control), 5% (w/v) HP-β-CD, and 15% (w/v) HP-β-CD. Afterthe HP-β-CD concentration adjustment, samples were left at 5° C.overnight. The following day, subvisible particle counts were measuredusing a HIAC 9703 particle counter.

The effect of 0%, 5% and 15% (w/v) concentration of HP-β-CD onresolubilization of SVP was tested. The results demonstrate that thereis a significant reduction in SVP in samples containing HP-β-CD relativeto the control sample (0% HP-β-CD). As shown in FIGS. 12A-12F, 15%HP-β-CD effectively resolubilizes particles greater than or equal to 1.4microns, whereas 5% HP-β-CD effectively resolubilizes particles greaterthan or equal to 2 microns.

These results establish that in addition to preventing enzymaticpolysorbate degradation and solubilizing the PS20 degradants that resultfrom enzymatic digestion of polysorbate 20, cyclodextrins can alsosolubilize the PS20 degradation products that result from oxidativedigestion of polysorbate 20.

Example 8: The Effects of HP-β-CD Concentration and the HP-β-CD:PS20Ratio on PS20 Degradation

To determine the effect of HP-β-CD concentration on PS20 degradation,samples containing 0.001, 0.01, 0.1, 1, 5, or 15% PS20 were digestedusing 15 μg/mL of PPL for 4.5 hours. As shown in FIG. 13, increasing theamount of HP-β-CD reduces PS20 degradation.

The effect of the HP-β-CD concentration at different PS20 concentrationson the enzymatic degradation of PS20 was assessed to identify optimalconcentrations for inhibition of enzymatic PS20 degradation. To evaluatethe dependence of PS20 degradation on HP-β-CD concentration, the PS20content was determined using RP-ELSD for triplicate samples containingdifferent HP-β-CD concentrations at various PS20 concentrations. Samplescontaining 0.005% (FIG. 14A), 0.02% (FIG. 14B), 0.1% (FIG. 14C), and0.4% PS20 (FIG. 14D) were digested using 15 μg/mL of PPL enzyme for 4.5hours at room temperature in protein-free samples containing noexcipient (control), 0, 0.5, 5, and 15% (w/v) HP-β-CD. PPL was added toeach of the treatment solutions at a ratio of 75 mg PPL per mg PS20,with an equivalent volume of buffer added to the control solutions todetermine the effect of HP-β-CD to polysorbate ratio on enzymaticdegradation. Digestion was stopped by heat inactivation in an 85° C.water bath for 30 minutes. Each sample was analyzed for PS20concentration using high performance liquid chromatography with aninline evaporative light scattering detector as described above. Sampleswere then placed at 5° C. overnight to allow for the formation ofparticles and placed on ice during analysis for visible and subvisibleparticles as described above.

TABLE 2 HP-β-CD to PS20 ratio [HP-β-CD]/[PS20] [HP-β-CD]/[PS20] PS20remaining (wt/wt ratio) (mole ratio) (Relative %) Std dev 0 0 39.5 0.50.4 41.4 1.25 1.1 41.2 3.4 2.5 2.2 44.0 5 4.35 65.3 21.5 12.5 10.9 86.47.7 25 21.8 90.3 6.6 37.5 32.6 106.7 6.8 50 43.5 98.6 6.6 100 87.0 93.99.3 150 130.5 108.3 7.1 250 217.6 101.9 2.4 750 652.7 104.2 1.4 1000870.2 104.3 10.9 3000 2610.6 112.4 15.1

The amount of cyclodextrin is required for complete inhibition ofenzymatic PS20 degradation depends on the concentration of PS20 (FIGS.13 and 14A-D). At lower PS20 concentration (e.g., 0.005% PS20 (FIG.14A), only 0.5% HP-β-CD is required to fully inhibit PS20 degradationwhereas 15% HP-β-CD is required for samples containing 0.1% PS20 (FIG.14C). Similarly, formation of sub-visible particles greater than 2, 5,or 10μ in diameter is dependent on the ratio of cyclodextrin topolysorbate. Samples containing 0.02% PS20 required 0.5% HP-β-CD topartially inhibit sub-visible particle formation and 15% HP-β-CD tocompletely inhibit sub-visible particle formation (FIGS. 15A-15C)

These results can be interpreted in the context of the HP-β-CD to PS20ratio (w/w) (FIG. 16 and Table 2). The PS20 data demonstrate thatsufficient HP-β-CD:PS20 (≥37.5 w/w) is required to inhibit enzymaticPS20 degradation (Table 2). The results suggest that the HP-β-CD to PS20ratio is important in determining PS20 degradation across broadconcentration ranges for PS20 and HP-β-CD.

The results also elucidate a possible mechanism of inhibition of PS20degradation by HP-β-CD. In this case, the amount of PS20 degradationvaries sigmoidally as the inhibitor (i.e., HP-β-CD) concentration isincreased at fixed substrate (i.e., PS20) concentration (FIG. 13). Thus,without being bound by theory, increasing the cyclodextrin concentrationmay effectively reduce the free substrate concentration in solutionwhich decreases the rate of PS20 degradation. Alternatively, it ispossible that the PS20 degradation rate is inhibited by thesubstrate-inhibitor complex.

Example 9: Polysorbate Degradation Under Antibody Storage Conditions

The impact of various protein molecule classes (e.g., monoclonalantibody (mAb), single-Fab antibody (sFAb), and bispecificantibody(BsAb)) was assessed on the ability of cyclodextrins to decreaseenzymatic PS20 degradation. The mAb, sFAb, and BsAb drug substancesamples were provided in their native formulations. The samples weredialyzed and conditioned into the target formulation of 20 mM histidineacetate at pH 5.5 with 0.02% PS20 to a final protein concentration of 20mg/mL. The control sample was prepared to contain 20 mM histidineacetate, pH 5.5, 0.02% PS20. Each of the mAb, sFAb, BsAb, and controlsamples were sub-aliquoted and adjusted using conditioning buffer tocontain different amounts (0%, 5%, and 15%) HPβCD. Samples were digestedwith 15 μg/mL PPL enzyme for 4.5 hours at room temperature, followed by30 minutes of heat inactivation at 85 C. Each sample was analyzed forPS20 concentration using high performance liquid chromatography with aninline evaporative light scattering detector. Samples were then placedat 5° C. overnight to allow for the formation of particles and wereanalyzed for visible and subvisible particles as described above.

The results demonstrate that each sample containing 0% HPβCD has adifferent amount of PS20 degradation. The results show that the 0% HPβCDsamples containing protein (FIGS. 17B-D) have higher amounts of PS20degradation relative to the control sample (FIG. 17A). Because theproteins are expressed in Chinese hamster ovary (CHO) or E. Coli cells,the protein samples may contain other impurities (i.e., lipases, etc.)that contribute to the total amount of PS20 degradation.

Although the 0% HPβCD protein-containing samples were observed to havehigher amounts of PS20 degradation, the results demonstrate that thereare comparable amounts of PS20 degradation observed in samplescontaining 5% and 15% HPβCD. These results demonstrate that HPβCD isequally effective at mitigating catalytic PS20 degradation for all themolecular formats evaluated even though they may have different amountsof impurities that catalytically degrade PS20 (FIGS. 17A-D). Thisestablishes that the presence of protein molecules and their nativeimpurity profiles do not significantly affect the cyclodextrin toprotein ratio that is necessary to mitigate PS20 degradation. Withoutbeing bound by theory, this finding suggests that the mechanism ofcatalytic inhibition involves cyclodextrin molecules directlyinteracting with the PS20, and not the enzyme that is degrading thepolysorbate. Otherwise, the protein-containing samples, which containadditional enzymes that degrade polysorbate would require more HPβCD tomitigate the PS20 degradation compared to the control. Thus, thecyclodextrin to PS20 ratio described herein should be broadly applicableto a wide range of formulations containing different protein moleculesand impurity profiles.

CONCLUSION

The studies performed show the ability of cyclodextrins to inhibit theenzymatic and oxidative degradation of polysorbates (PS20 and PS80). Theresults demonstrate that PVP and cyclodextrins (i.e., HP-α-CD, HP-β-CD,HP-γ-CD, SBE-β-CD) were able to prevent enzymatic degradation of PS20.Further experiments demonstrate that HP-β-CD is protective ofpolysorbate in the presence of multiple enzymes (i.e., CALB, RLE, LPL,and PLL). Without being bound by theory, the inhibitory mechanism mayinvolve an interaction between the inhibitor (i.e., cyclodextrin) andthe substrate (i.e., polysorbate). The inclusion complex formation mayboth reduce the concentration of free substrate and the inclusioncomplex may also be directly sterically inhibiting the interaction withthe active site and the substrate. Additionally, concentration studiesestablish that there is an optimal range of HP-β-CD to PS20 ratio (≥37.5w/w) that is necessary to provide complete inhibition of enzymatic PS20degradation.

In addition to preventing enzymatic PS20 degradation, the resultsdemonstrate that cyclodextrins can effectively reduce the amount ofsubvisible and visible particles. In this manner, cyclodextrinsdisaggregate and dissolve subvisible and visible particles in solution.In addition to effectively preventing the formation of particles, theresults demonstrate that cyclodextrins also can effectively solubilizeexisting particles related to polysorbate degradation. Presumably,cyclodextrins also increase the solubility of free fatty acids that areproducts of polysorbate degradation.

The findings from this study have extensive practical implications. Theresults provide comprehensive evidence that formulation containingcyclodextrins may be used to prevent enzymatic polysorbate degradation.Additionally, cyclodextrins can be used to solubilize free fatty acidsassociated with polysorbate degradation from both oxidative andenzymatic degradation. Thus, cyclodextrins may also be useful asdiluents or reconstitution buffers for drug products to dissolvedegradants and particles associated with polysorbate degradation.

1-4. (canceled)
 5. A method to disaggregate and solubilize polysorbatedegradation products in an aqueous formulation comprising a polysorbate,the method comprising adding a cyclodextrin to the formulation, whereinthe resulting w/w ratio of cyclodextrin to polysorbate is greater thanabout 37.5:1. 6-27. (canceled)
 28. An aqueous formulation comprising apolysorbate and a cyclodextrin, wherein the formulation has been storedat about 1° C. to about 10° C. for at least about six months, whereinthe initial w/w ratio of cyclodextrin to polysorbate in the formulationis at least about 37.5:1 and wherein the amount of polysorbate in theformulation is at least about 80% of the initial amount of polysorbatein the formulation.
 29. An aqueous formulation comprising a polysorbateand a cyclodextrin, wherein the formulation has been stored at about 1°C. to about 10° C. for at least about six months, wherein the w/w ratioof cyclodextrin to polysorbate in the formulation is at least about37.5:1 and wherein less than about 1% of the polysorbate has degraded.30. The formulation of claim 28, wherein the cyclodextrin isHP-βcyclodextrin, HP-γcyclodextrin, or sulfobutyl ether β-cyclodextrin.31. The formulation of claim 28, wherein the concentration ofpolysorbate in the formulation is in the range of about 0.01% to 0.4%about 0.01% or 0.1%, or about 0.02%. 32-33. (canceled)
 34. Theformulation of claim 28, wherein the concentration of cyclodextrin inthe formulation is in the range of about 0.5 to 30% or is about 15%. 35.(canceled)
 36. The formulation of claim 28, wherein the polysorbatedegradation is reduced by about 50%, about 75%, about 80%, about 85%,about 90%, about 95% or about 99%.
 37. The formulation of claim 28,wherein less than about 1,000, about 750, about 500, about 250, about150, about 100, about 50, or about 25 polysorbate particles greater thanabout 2 microns in diameter/mL are formed.
 38. The formulation of claim28, wherein the formulation further comprises a polypeptide.
 39. Theformulation of claim 38, wherein the polypeptide is an antibody.
 40. Theformulation of claim 39, wherein the antibody is a polyclonal antibody,a monoclonal antibody, a humanized antibody, a human antibody, achimeric antibody, a multispecific antibody or an antibody fragment. 41.The formulation of claim 28, wherein the polypeptide concentration inthe formulation is about 1 mg/mL to about 250 mg/mL.
 42. The formulationof claim 28, wherein the formulation is stable at about 2° C. to about8° C. for at least about six months.
 43. The formulation of claim 28,wherein the formulation is stable at about 1° C. to about 10° C. for atleast about forty-eight months or is stable at about 2° C. to about 8°C. for at least about forty-eight months.
 44. (canceled)
 45. Theformulation of claim 28, wherein the formulation has a pH of about 4.5to about 7.0, about 4.5 to about 6.0, or about 6.0. 46-47. (canceled)48. The formulation of claim 28, wherein the formulation furthercomprises one or more excipients selected from the group consisting of astabilizer, a buffer, a surfactant, and a tonicity agent.
 49. Theformulation of claim 28, wherein the formulation is a pharmaceuticalformulation suitable for administration to a subject.
 50. Theformulation of claim 28, wherein the formulation is a pharmaceuticalformulation suitable for intravenous, subcutaneous, intramuscular, orintravitreal administration to a subject.